Treatment of skin conditions

ABSTRACT

The present invention provides piperine and analogues or derivatives thereof for the treatment of skin conditions treatable by stimulation of melanocyte proliferation, such as vitiligo, and also for treating skin cancer. The piperine and analogues or derivatives thereof may also be used to cosmetically promote or enhance the natural coloration of the skin.

REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part application from PCT(U.S.) application GB 99/02256 (Publication N⁰ WO 00/02544) filed Jul.13, 1999.

FIELD OF THE INVENTION

[0002] This invention relates to the treatment of skin conditions,comprising those conditions requiring stimulation of melanocyteproliferation and to the inhibition of melanomas. The invention is ofespecial application to the treatment of vitiligo and skin cancer.

[0003] Vitiligo is a common skin pigment disorder characterised by thedevelopment of patchy de-pigmented lesions. Current treatments whichinclude the use of photosensitisers (eg psoralens) with UVA radiation(PUVA), corticosteroids or skin grafting have low success rates and aregenerally accompanied by unpleasant side effects. Vitiligo has a highlydetrimental impact on the emotional well-being of the sufferer, thedisfiguring effects of the disease being compounded by the absence of asuitable treatment. Although vitiligo patches are not believed tocontain melanocytes (pigment producing cells), a reservoir exists inhair follicles in vitiliginous skin. Thus activation of hair follicularmelanocytes is a crucial process in the repigmentation of vitiliginousskin.

[0004] Certain plant remedies, usually administered as mixtures of herbsor extracts, particularly those used in traditional Chinese medicine andIndian Ayurvedic medicine, have been employed for the treatment ofvitiligo for a long time and in many cases have given positive resultsin small scale studies. Herbs such as Psoralea corylifolia L andVernonia anthelmintica Willd. (=Centratherum anthelminticum Kuntze) arewell known for their use in this disease. Psoralens, which are employedin the modem PUVA and khellin in KUVA therapy were originally derivedfrom plant sources (Psoralea corylifolia L and Ammi visnagarespectively) used in traditional remedies for vitiligo. However thesetherapies rely on the use of UV irradiation for their efficacy, which isassociated with the aetiology of skin cancer.

[0005] The fruit of black pepper (Piper nigrum L.) and long pepper(Piper longum L.) are both important medicinal herbs in Ayurvedic andUnani (traditional Indian) medicine systems, in which remedies generallyconsist of mixtures of herbs. A wide range of the medicinal uses ofblack pepper have been documented by Kirtikar and Basu (Indiam MedicinalPlants, 2^(nd) Edition, Vol. 3, (1935) pages 2128-2135), including itsuse in the treatment of leucoderma. Black pepper has also beenimplicated as a possible adjunct to Vernonia anthelmintica in thetreatment of leucoderma (Indian Medicinal Journal, Vol. 1, 3^(rd)Edition, (1982) 1267-1270). These two herbs are employed as aconstituent in many traditional herbal preparations for a variety ofuses, including gastrointestinal and skin ailments. Compositionscomprising black pepper, ginger and pipali have been used in thetreatment of vitiligo (Ancient Science of Life, Vol. IX, No. 4 (1990)202-206); however, the specific therapeutic action of black pepper inthis orally administered composition has not been established.

[0006] There is, therefore, a need for further compounds andcompositions, which are able to stimulate the proliferation ofmelanocytes.

SUMMARY OF THE INVENTION

[0007] It has been surprisingly found that, piperine, which is presentin the fruit of Piper nigrum, stimulates the replication of melanocytes.The action of piperine is to increase the number of cells which conferpigmentation. Piperine is the compound(E,E)-1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine andshould not be confused with piperidine.

[0008] Piperine has also been reported to occur in other Piper speciesie. P. acutisleginum, album, argyrophylum, attenuatum, aurantiacum,betle, callosum, chaba, cubeba, guineense, hancei, khasiana, longum,macropodum, nepalense, novae hollandiae, peepuloides, retrofractum,sylvaticum. Pharmaceutical compositions containing piperine have beenused in the treatment of tuberculosis and leprosy (EP 0 650 728). It hasalso been suggested that piperine is able to enhance the bioavailabilityof the other constituents of a pharmaceutical composition (WO 96/25939).

[0009] The invention provides a method of treating a subject (human oranimal) having a skin condition requiring stimulation of melanocyteproliferation and melanomas, which comprises administering to thesubject, preferably to the site of the condition, an effective amount ofpiperine or an active analogue or derivative thereof, as hereinafterdefined.

[0010] The active ingredient may be used on its own, but is moresuitably used in combination with a carrier or excipient and optionallyone or more further active ingredients. It may also be used in the formof an isolate or plant extract, in the case of piperine itself derivablefrom Piper nigrum, for example.

[0011] Stimulation of melanocyte proliferation greatly facilitates there-pigmentation of de-pigmented skin, e.g. post traumatised de-pigmentedskin. The term “post traumatised de-pigmented skin” means the skinformed during the healing process that occurs after a skin trauma.De-pigmentation may arise, for example, from scar tissue formed as aresult of a skin trauma such as burn or other skin lesion or may be dueto vitiligo. The present invention can be used to treat any of theseskin disorders in a patient.

[0012] Generally in this invention, the piperine or active derivative oranalogue thereof may be administered by oral, topical, intravenous orsubcutaneous (intra-muscular) routes but is preferably applied topically(to the area of the skin where treatment is desired).

[0013] The active ingredient may be formulated as a solid powder; apaste, ointment or cream; a tablet or capsules; or a solution.

[0014] The method of the invention may also be used to treat a personhaving a skin condition which would benefit from coloration, e.g. toenhance or promote the natural colouring of the skin. The treatment maybe used for prophylactic, therapeutic or cosmetic purposes.

[0015] Piperine and its analogues or derivatives as hereinafter definedinhibit the proliferation of melanoma cells. Thus, they may also be usedin the treatment of skin cancer. Another aspect of the inventiontherefore provides a method of treating skin cancer in a human or animalpatient comprising the administration to said patient of atherapeutically effective amount of piperine or an active analogue orderivative thereof, as hereinafter defined.

[0016] The piperine or active analogue or derivative thereof may beadministered by oral or topical routes. Suitable dosage forms may be anyof those discussed above.

[0017] The formula of piperine and derivatives and analogues thereofusable in this invention is given below.

[0018] wherein

[0019] n=0 or 1;

[0020] p is 0 or 1;

[0021] q is 0 or 1

[0022] when n=p=q=0, R³ and R⁴ represent hydrogen or together representa carbon to carbon double bond;

[0023] when n=0 and one of p and q=1, R³ and R⁴ together and one of R⁵and R⁶ together or R⁷ and R⁸ together represent carbon to carbon doublebonds, R³ and R⁴ together represent a carbon to carbon double bond andR⁵ and R⁶ or R⁷ and R⁸ represent hydrogen atoms, R³ and R⁴ representhydrogen and one of R⁵ and R⁶ together or R⁷ and R⁸ together representcarbon to carbon double bonds or R³, R⁴, R⁵, R⁶, R⁷ and R⁸ all representhydrogen atoms;

[0024] when n=0 and p=q=1, R³ and R⁴ together and one of R⁵ and R⁶together or R⁷ and R⁸ together represent carbon to carbon double bondsthe other of R⁵, R⁶, R⁷ and R₈ representing hydrogen, R³ and R⁴ togetherrepresent a carbon to carbon double bond and R⁵ and R⁶ or R⁷ and R⁸represent hydrogen atoms, R³ and R⁴ represent hydrogen and one of R⁵ andR⁶ together or R⁷ and R⁸ together represent carbon to carbon doublebonds the other of R⁵, R⁶, R⁷ and R⁸ representing hydrogen, R³ and R⁴together, R⁵ and R⁶ together and R⁷ and R⁸ together represent carbon tocarbon double bonds or R³, R⁴, R⁵, R⁶, R⁷ and R⁸ all represent hydrogenatoms;

[0025] or optionally when n is 1 R² and R³ together represent a carbonto carbon double bond and one or more of R⁴ and R⁵ together, R⁵ and R⁶together, R⁶ and R⁷ together or R⁷ and R⁸ together represent a carbon tocarbon double bond the other of R⁴ to R⁸ representing hydrogen;

[0026] m=1, 2 or 3;

[0027] when m=1, R¹ represents an alkoxy group having from 1 to 3 carbonatoms or a hydroxy group;

[0028] when m=2, each R¹ independently represents an alkoxy group havingfrom 1 to 3 carbon atoms or the two R¹s together represent a3′,4′-methylenedioxy group;

[0029] when m=3, two R¹s together represent a 3′,4′-methylenedioxy groupand the other R¹ represents an alkoxy group having from 1 to 3 carbonatoms or a hydroxy group;

[0030] R⁹ represents a pyrrolidino, piperidino, 4-methylpiperidino ormorpholino group, a N-monoalkylamino group of 4 to 6 carbon atoms, aN-monocycloalkylamino group of 4 to 7 carbon atoms, a3′,4′-methylenedioxy-substituted benzylamino or 2-phenethylamino groupor R⁹ represents an alkoxy group of 1 to 6 carbon atoms; in any of itsE, Z geometrically isomeric forms.

[0031] Certain of the active analogues or derivatives of piperine offormula (1) are new. The present invention therefore includes suchcompounds, and pharmaceutical compositions containing them together witha carrier or excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1: Plots of growth of melan-a-cells cultured in differentmedia. Each point represents the mean and standard deviation (SD) of 6replicates. FBS=fetal bovine serum. TPA=tetradecanoyl phorbol acetate.

[0033]FIG. 2: Plots of growth melan-a-cells cultured from differentinitial plating densities of cells. Medium was supplemented with 20 nMTPA. On day 4 the medium in the remaining plates was replaced. Eachpoint shows mean and SD of 6 replicates.

[0034]FIG. 3: Effect of P. nigrum extract on the growth of melan-acells. Culture was maintained for 8 days. Medium and extract werereplaced with fresh ones on day 4. Each point designates mean and SD of6 replicates, except that 12 replicates were done for cells only.

[0035]FIG. 4: Effects of P. nigrum extract and TPA on the proliferationof melan-a cell line. Each point shows mean and SD of 6 replicates,except that 12 replicates were done for cells only.

[0036]FIG. 5: Effects of piperine and TPA on the growth of melan-a cellsin the presence of RO-31-8220. n=6 for piperine and TPA treated wells,whereas n=12 for RO-31-8220 alone.

[0037]FIG. 6: Effects of piperine and TPA on the growth of humanmelanoblasts in the presence of ET3. *P<0.05 when compared to vehiclecontrol (One way Anova, followed by Dunnett's t-test).

[0038]FIG. 7: Effects of piperine on the growth of human melanocytes inthe presence of ET1. *P<0.05 when compared to ET1 1 nM treatment (Oneway Anova, followed by Dunnett's t-test).

[0039]FIG. 8: Dose response curve showing the growth of melan-a cells inpresence of a compound of formula (1), RV-A01, as % of control plottedagainst concentration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] One useful class of compounds of formula (1) is that in which

[0041] (a) n is 0, p and q are each 0 or 1, m is 2, the R¹s togetherrepresent a 3′,4′-methylenedioxy group, R³ and R⁴, together with thecarbon atoms to which they are attached form a carbon to carbon doublebond and, when p and q are each 0 or 1, R⁵ and R⁶ and R⁷ and R⁸ togetherwith the carbon atoms to which they are attached, form a carbon tocarbon double bond and R⁹ is piperidino, or

[0042] (b) n is 0, one of p or q is 1 and (i) m is 3, the R¹s being3′,4′-methylenedioxy and 6′-methoxy or (ii) m is 2, the R¹s being3′-hydroxy-4′-methoxy; or (iii) m is 1 and the R¹ is 4′-hydroxy; and R³to R⁹ are as defined in case (a) above, or

[0043] (c) n is 0, one of p and q is 1, R⁹ is piperidino, pyrrolidino,isobutylamino or methoxy and all other symbols are as defined in case(a) above, or

[0044] (d) n is 0, one of p and q is 1, R⁵, R⁶, R⁷ and R⁸ representhydrogen atoms and either R³ and R⁴ also do or R³ and R⁴ together withthe carbon atoms to which they are attached form a carbon to carbondouble bond; and m, R¹ and R⁹ are as defined in case (a) above;

[0045] (e) n is 0, p q=1 and R³, R⁴, R⁵, R⁶, R⁷ and R⁸ representhydrogen;

[0046] (f) n is 0, one of p and q is 1, R³, R⁴, R⁵, R⁶, R⁷ and R⁸represent hydrogen and R⁹ is cyclohexylamino; and

[0047] in all of which cases (a) to (f) the molecule is in the E,E orall E geometric configuration or in case (a) when n is 1 may be in theZ,Z, Z,E or E,Z geometric configuration.

[0048] The following are preferred features of the compounds of formula(1) considered alone or in any possible combination of two or more:

[0049] n is 0, one of p and q is 1, R³ and R⁴ together and one of R⁵ andR⁶ together or R⁷ and R⁸ together represent double bonds or R³, R⁴, R⁵,R⁶, R⁷ and R⁸ all represent hydrogen atoms

[0050] m is 2 or 3, two R¹s represent 3′,4′-methylenedioxy andoptionally a third R¹, representing 6′-methoxy, is also present

[0051] R⁹ represents a piperidino, 4-methylpiperidino, pyrrolidino ormorpholino group or an alkylamino group having 4 to 6 atoms, preferablybranched chain and especially an isobutylamino (2-methylpropylamino)group, a cycloalkyl amino group of 4 to 7 carbon atoms, especially acyclohexylamino group, or a 3,4-methylenedioxy-substituted benzylaminoor 2-phenethylamino group

[0052] alternatively R⁹ is an alkoxy group having from 1 to 6 carbonatoms, preferably 3 to 6

[0053] the geometric configuration at the double bonds is as in piperine(all E, E)

[0054] While the preferred meaning of R¹ is a 3′,4′-methylenedioxygroup, R¹ may alternatively be provided by one, two or even three groupsselected from hydroxy and alkoxy of 1 to 3 carbon atoms, preferablymethoxy, e.g. as in 3′-methoxy, 4′-methoxy, 6′-methoxy and3′,4′-dimethoxy substitution of the left-hand benzene ring.

[0055] The twice-daily topical application of compounds of formula (I)has been found to induce significant pigmentation in mice. Skincoloration in the mouse population under study was first observed atapproximately four weeks after the treatment was started. Thiscoloration was enhanced further as a result of subsequent topicalapplications.

[0056] Specific preferred compounds for use in the invention are asfollows:

[0057] Variations and alterations (all other structural features of themolecule are as in piperine unless otherwise indicated) Compounds offormula (1) and trivial names Variation in stereochemistry at doublebonds 1 (E, E) - Piperine and in extent of conjugation in chain 2 (Z,Z) - Chavicine

3 (Z, E) - Isopiperine 4 (E, Z) - Isochavicine 5 3,4-dihydropiperine -Piperanine 6 1,2,3,4-tetrahydropiperine Variation in separation of rings(conjugated) Structures (all E)

7 n = 0 - Ilepcimide 1 n = 1 - Piperine 8 n = 2 - PiperettineAlterations to nitrogen substituent Structures (all E, E)

R⁶ =1 piperidino - Piperine 9 pyrrolidino - Trichostachine 10isobutylamino - Piperlonguminine 11 methoxy -Despiperidylmethoxypiperine 17 morpholino 18 hexylamino 193′,4′-methylenedioxybenzylamino Alterations to the phenyl substituentStructures (all E, E)

1 3′,4′-methylenedioxyphenyl; n = 1 - Piperine 12 As 1 + 6′-methoxy; n =1; - Wisanine 13 3′-hydroxy,4′-methoxyphenyl; n = 1 -4′-Methoxyisocoumaperine 14 4′-hydroxyphenyl; n = 1 20 4′-methoxyphenyl;n = 0 Alterations to connecting chain and amide group

21 cyclohexylamino; n = 1 22 cyclohexylamino; n = 0

[0058] The naturally occurring compounds (including piperine) can beextracted from suitable plant sources or synthesised using methods knownto a skilled person (see, for example, Chapman and Hall, CombinedChemical Dictionary on CD-Rom, Release 1:1 (1997) and The Merck Index(1983), 10th edition. Publ. Merck and Co, Rahway, USA. PP. 1077-1078(except compounds 2 and 3)). Many of the above, occur in P. nigrum orother Piper species (10 and 12).

[0059] Compounds 2 and 3 can be prepared by isolation from P. nigrumusing methods known to a skilled person (see, for example, Cleyn R Deand Verzele M (1975). Constituents of Peppers. Part VII. SpectroscopicStructure Elucidation of Piperine and its Isomers. Bulletin de laSociete Chimique Belgique, 84, 435-438).

[0060] Compound 6 can be prepared by hydrogenation of piperine, usingknown methods.

[0061] Compound 11 can be prepared by methanolysis of piperine usingsodium methoxide.

[0062] Compound 13 can be prepared from 3-hydroxy-4-methoxybenzaldehydeusing methods analogous to those used for the preparation of piperine.

[0063] Other compounds within formula (1) can be prepared from theappropriate acid with the appropriate connecting chain between thecarboxylic acid function and the benzene ring and having the appropriatestereochemistry. Where necessary, this may be preceded or followed byreduction to reduce the double bond or bonds in the connecting chain.Methods of preparing amides and esters from these acids are illustratedby the Examples below. They may also be adapted from the referencescited herein, the disclosure of which is herein incorporated byreference.

[0064] The active compounds may be formulated for topical use in theform of creams, soft paraffin or lotions. Aqueous cream BP or YellowSoft Paraffin BP may suitably contain the active at 0.03-3.0 mg % w/w oran equivalent amount of plant extract. A suitable lotion is typicallyprepared from 20% glycerol and 80% ethanol in purified water andcontains 0.03-3.0 mg % w/w of the active material. These topicalformulations may also contain penetration enhancers such as oleic acid,propylene glycol, ethanol, urea, lauric diethanolamide or azone,dimethyl sulphoxide, decylmethyl sulphoxide, or pyrrolidone derivatives.Liposomal delivery systems may also be used.

[0065] Compositions for oral formulation include tablets or capsulescontaining 1.5-150 mg active or equivalent amount of plant extract.

[0066] The invention will now be described with reference to thefollowing non-limiting examples, with reference to the accompanyingtables and drawings.

EXAMPLES

[0067] Plant samples and preparation of extracts

[0068]Piper nigrum L. fruit (black pepper, Piperaceae), originally fromIndia, was purchased from the Food Centre, 70 Turnpike Lane, London N8,UK. The rest of the herbs were either supplied by East-West Herbs,Kingham, Oxon, UK or by Cipla Ltd, Mumbai, India.

[0069] For the preliminary screening programme, the powdered dry herb(10 g) was heated to boiling in distilled water (100 ml) and allowed toboil for 10 min, using a hot plate as heat source. The plant materialwas filtered off under vacuum through filter paper (Whatman), and thefiltrate freeze-dried.

Cell Culture Experiments

[0070] Microplate culture and sulforhodamine B (SRB) assay

[0071] Cells of mouse melan-a cell line (passage number 18-24), a firstknown line of non-tumorigenic pigmented mouse melanocytes weremaintained in a flask (Costar, Cambridge, Mass., USA) using RPMI 1640(ICN, Costa, Mesa, Calif., USA) as a basic medium. For microplateproliferation assays, subconfluent melan-a cultures were trypsinized(0.25% trypsin at 37° C. for 5-10 min) and inoculated with arepeater-pipettor (Finn pipette, Labsystems, Finland) into 96-wellmicrotiter plates (Costar, Cambridge, Mass., USA). The plates wereincubated at 37° C. in a 10% CO₂, 90% air humidified atmosphere for thestated length of time. At the end of the incubation, an SRB assay wasperformed. Briefly, cells attached to the bottom of the plate were fixedby addition of cold trichloroacetic acid (TCA, 4° C., Aldrich, Dorset,UK) on the top of the growth medium (final TCA 20% w/v). The plate wasplaced at 4° C. for 1 hour before being gently washed five times withtap water. It was allowed to dry in air, or aided with a hair dryer tospeed up the drying process, then 50 μl of 4% w/v SRB dissolved in 1%acetic acid in water was added to each well for 30 min. At the end ofthe staining period, unbound SRB was removed by washing 4 times with 1%acetic acid. The plate was air dried again, and 150 μl of 10 mM aqueousTris base (Sigma-Aldrich Co. Ltd, Irvine, UK) was added into each wellto solubilize the cell-bound dye. The plate was shaken for 15 min on agyratory shaker followed by reading the optical density (OD) at 550 nmin a microplate spectrophotometer (Anthos Labtec HT3, version 1.06)

Example 1

[0072] Optimisation of incubation conditions—FBS concentration and cellseeding density

[0073] Prior to testing the herbal extracts, optimal culture conditionswere established. The variable factors regarding incubation conditionsinclude foetal bovine serum (FBS) concentration, initial cell seedingdensity and incubation period. To determine optimum FBS concentration,1, 2, and 5% FBS were used to culture the melan-a cell line, the growthpattern with each concentration of FBS was monitored by SRB assay. Forthe determination of optimum cell seeding density, a series of initialseeding density of 0.15 to 1.2×10⁴ cell per well of melan-a cells wereplated into 96-well plates with 5% FBS and 20 nM tetradecanoyl phorbolacetate (TPA) supplemented growth medium. The growth pattern wasmonitored with SRB assay at daily intervals. The culture was extended to8 days; on day 4, the medium in the remaining plates was replaced.

Results

[0074] The effect of FBS concentrations on melan-a growth

[0075] The optimal condition for the negative experimental control, isthat cells neither grow too fast nor decline dramatically. Rapid growthmight mask any subtle stimulatory effect brought about by the herbalextracts, whereas a dramatic decline in cell numbers indicatesunfavourable culture conditions for cell survival, which could lead tocell damage. FIG. 1 shows the growth curves of melan-a cell line atthree different concentrations of FBS. Neither 1% nor 2% FBSsupplemented medium was able to maintain cell survival; cell numbersdeclined significantly in 4 days of culture. However, 5% FBS was capableof keeping melan-a cell line alive with only a small increase in cellnumbers observed over 4 days. TPA (20 nM) was able to cause furtherproliferation in the presence of 5% FBS indicating that cells werecapable of responding to mitogenic stimuli at 5% FBS. Morphologicalobservations under a microscope revealed that with 1% and 2% FBSsupplemented medium, cell bodies were round, lightly pigmented with fewdendritic processes and the culture displayed an ageing growth pattern.However in 5% FBS, cells possessed more melanosomes and some shortdendrites without an ageing appearance. Therefore 5% FBS was usedthroughout in the herbal screening experiments.

[0076] Growth curves of melan-a cell line with various seeding densities

[0077] In FIG. 2, growth curves over 8 days with different initial cellnumbers were plotted to elucidate the melan-a cell line's growth patternin 96-well plates in the presence of 5% FBS and 20 nM TPA. The optimalinitial plating density together with proper harvesting time wasdetermined. All of the initial plating number of cells showed a netgrowth in the presence of TPA and 5% FBS supplemented medium, althoughthe higher plating density of 1.2×10⁴ cells/well depleted the growthmedium on day 3 of culture and the cells ceased to grow until the mediumwas replaced. With the lower plating densities (2-4×10³ cells/well) theSRB assay OD readings remained relatively low after 8 days' culture. Theinitial plating density of 6×10³ cells/well exhibited exponentialgrowth, and after 4 days of culture, the OD reading increased to a valueof about 0.4. Since the higher OD values are associated with greaterprecision and accuracy, it was determined that the initial inoculationof 6×10³ cells/well was the optimum density for the herbal testexperiment. For the simplicity of the experiment, harvesting time wasday 4 since the cells at this stage was not confluent and after 4 days,growth medium tended to become depleted and replacement was necessaryfor the further growth.

Example 2

[0078] Preliminary herbal screening experiments

[0079] Melan-a cells were seeded at a density of 6×10³/100 μl/well instandard medium supplemented with 0 nM TPA and 5% FBS. After 4 hours ofincubation, herbal extracts, which were reconstituted in growth mediumand sterilised by filtration (pore size 0.2 μm), of differentconcentrations was added into each well. Final concentrations of plantextract were 0 (negative control), 10, 100 and 1000 μg dry extract perml. 6 replicate wells were used for each concentration tested. Thenegative control (12 wells), positive control (20 nM TPA, 6 wells), andtest wells were all in the same 96-well plate. The culture wasterminated after 4 days and SRB assay performed according to the methodsgiven above.

Results

[0080] The effect of 30 herbal extracts on the proliferation of melan-acell line

[0081] Table 1 shows the results of the preliminary screening of 30aqueous herbal extracts on the proliferation of melan-a cell line. Crudeextracts of Astragalus membranaceous (Fisch.) Bunge, unripe Citrusreticulata Blanco, Dictamnus dasycarpus Turcz., Ophiopogon japonicus(Thunb.) Kergawe, Piper nigrum L., Poria cocos (Schw.) Wolf and Tribulusterestris L. were observed to stimulate melanocyte proliferation,sometimes even at the lowest dose level of 10 μg/ml. Other extractseither had no significant effect or were cytotoxic. Among these positiveresponses, that of Piper nigrum L. extract at 0.01 and 0.1 mg/ml was themost pronounced. Piper nigrum extract at these two concentrations notonly strikingly enhanced cell growth, but this extract also altered thecell morphology. In the presence of Piper nigrum extract, the cellularbodies were smaller, with more and longer bipolar or polydendriticprocesses, an effect similar to that observed with TPA.

Example 3

[0082] Repeats of the tests on Piper nigrum extract on the melan-a cells

[0083] A newly prepared Piper nigrum fruit extract was tested on a newbatch of melan-a cell line with the culture in microplates extended to 8days. The effects of Piper nigrum extract on the growth of melan-a cellline were evaluated by SRB assay.

Results

[0084] Repeats of the tests of Piper nigrum extract on melan-a cells

[0085] In the light of the positive results from the preliminaryexperiment, further investigations on Piper nigrum extract were carriedout. FIG. 3 shows that the result of the significant proliferant effectbrought about by the Piper nigrum extract was even more marked on theextension of the incubation period to 8 days of culture, the growth was272% of the control (cells only). Microscopically, the morphology of thecells was altered as those seen in the preliminary experiments.

Example 4

[0086] Confirmation of the proliferant effect of Piper nigrum byhaemocytometer counting

[0087] Melan-a cells were plated in petri dishes (Ø35 mm, Nunclon,Denmark) with a plating density of 2×10⁴/ml and Piper nigrum extract atconcentrations of 0.01 and 0.1 mg/ml. A negative control (cells inmedium only) and positive TPA (20 nM) control were also set up. After 4days the cells in each dish were harvested and counted withhaemocytometer.

Results

[0088] Confirmation of the proliferant effect of Piper nigrum byhaemocytometer counting

[0089] SRB assay indirectly estimates cell number through proteinstaining and spectrophotometric measurement. To confirm if Piper nigrumextract stimulates melan-a cell proliferation, a direct cell countingwith haemocytometer method was employed. Table 2 shows the cell numbersin the presence of Piper nigrum extract and 20 nM TPA. Cell number underthe influence of Piper nigrum extract at 0.01 and 0.1 mg/ml wereincreased significantly compared to control, but less than that with 20nM TPA. This result is consistent with the finding in 96-well microplateSRB assay.

Example 5

[0090] Effect of Piperine on the growth of melan-a cell line

[0091] Piperine (Sigma-Aldrich Co. Ltd, Irvine, UK) was dissolved inMeOH, sterilised by filtration through a membrane (pore size 0.2 μm) anddiluted with standard growth medium. The final concentrations in culturewere 0.1 and 1 μM. A separate experiment (data not shown) showed thatthe concentration of MeOH present in these experiments was not toxic orproliferant to the cells.

Results

[0092] The effect of piperine on the proliferation of melan-a cell line

[0093] The effect of this compound on melan-a cell line is shown in FIG.4. Piperine at the two concentrations tested significantly stimulatedmelan-a proliferation. This compound brought about morphologic changesto melan-a cells, with smaller cell bodies, more and longer cellulardendrites, resembling those alterations induced by Piper nigrum extractand TPA. This indicates that piperine is an active principle responsiblefor the observed proliferant effect of Piper nigrum.

Example 6

[0094] Test of piperine on different cell types to determine itsspecificity

[0095] In order to determine the specificity of piperine, a panel ofdifferent cell types were employed to facilitate this investigation.These included melan-a, melan-c, SVK14, CSM, XB2, SC1, B16F10, IM9,CACO2, Swiss 3T3 cell lines and normal human lymphocytes. TPA (20 nM)was also tested on these cells. Table 3 shows the biological origin ofthe cells and an outline of the cell culture protocols.

Results

[0096] The effects of piperine and TPA on the growth of a panel of celltypes.

[0097] From Table 4, it can be seen that piperine has a highly selectiveeffect on the growth of a panel of cell types, since it only stimulatesthe mouse melanocytes (melan-a, melan-c), human melanoblasts (FM21E),human foetal melanocytes (FM 21E) and the mouse fibroblast SC1 celllines at the concentration tested. The SC1 cell line may have aparticular sensitivity to TPA due to the way in which it has beenderived, i.e. it has been cultured in the presence of TPA. However,piperine has either no effect or a cytotoxic effect on other cells. Thisresult implies that piperine may have desirable specificity index forthe proliferation of melanocytes in culture and is not a generalmitogen. In our experimental system, TPA, a well known PKC activator anda tumour promoting agent, had similar effects to piperine on all celltypes tested, except that TPA strikingly stimulated human lymphocyte and3T3 fibroblast proliferation whereas piperine obviously lacked such anactivity. Piperine seems to be a less potent stimulant than TPA.

Example 7

[0098] Mode of Action: effect of RO-31-8220 on the growth of melan-acells with piperine and TPA

[0099] Melan-a cell line cultured with piperine 1 μM and TPA 20 nMseparately was set up in a 96-well plate, 1 μl of differentconcentrations of RO-31-8220 (Calbiochem-Novabiochem) in DMSO wasintroduced with a micro-syringe into the wells to make up the finalRO-31-8220 concentrations of 0 (control), 0.1, 1, 5, 10, 100 nM, withfinal DMSO concentrations smaller than 0.01% v/v, at which the DMSOshowed neither toxic nor proliferant effect to the cells in a separateexperiment (data not shown). 6 replicate wells were used for eachconcentration. The culture was incubated for 4 days before it wasterminated and processed with SRB assay to evaluate the growth ofmelan-a cells.

Results

[0100] Mode of Action: effect of RO-31-8220 on the growth of melan-acells with piperine and TPA

[0101]FIG. 5 shows the effect of RO-31-8220 on the survival and growthof melan-a cell line in the presence or absence of piperine and TPA.RO-31-8220 alone did not have significant cytotoxic effect to the cellsat the concentrations up to 100 nM. However, the proliferant effects ofpiperine, and TPA (as indicated by the Y axis values) on melan-a cellswere effectively inhibited by the presence of RO-31-8220 at theconcentrations of 0.1-100 nM. It thus appears that piperine and TPAexert their proliferant effects through the activation of PKC cellsignalling pathway.

[0102] The selectivity of piperine on the growth of a panel of celltypes has also been tested. It was found that piperine possessed afairly high specificity and selectivity towards melanocytes, since itsignificantly stimulated the growth of melan-a, melan-c and FM21Emelanoblasts and FM21E melanocytes in culture, whereas it did notstimulate all other cells apart from a TPA-sensitive fibroblast cellline. Piperine was observed to have inhibitory effects on B16 mousemelanoma cell line which is syngeneic with melan-a cells. Thus piperinemay be a specific stimulant for the proliferation of melanocytes invitiliginous skin without the risk of stimulating melanoma cells.

Example 8

[0103] Experiments on human melanoblasts in culture

[0104] Human melanoblasts in culture in this experiment were establishedfrom human foetal skin. Subconfluent melanoblasts maintained in MCDB 153medium supplemented with 10% FBS, 10 ng/ml stem cell factor (SCF) and 1nM endothelin 3 were subcultured and inoculated into 96-well microplatewith 6×10³ cells/100 μl/well. After incubation in the 10% CO₂,humidified atmosphere, at 37° C. for 3-4 hours to allow the attachmentof the cells on the plate, piperine dissolved in MeOH and water wasadded into the wells. The final concentrations of piperine were 1, 5,10, 100 μM, with TPA (20 nM) as positive control. Six replicates wereused in each group of treatment, with 12 wells used for vehicle control.The incubation was conducted for 5 days before cells were harvested byfixing with cold trichloroacetic acid (TCA, at 4° C., finalconcentration 20% v/v), and evaluated for cell number using an SRBassay. One way ANOVA and Dunnett's t-test was employed to test thesignificance of any differences between treatment groups and vehiclecontrol. Growth in the presence of piperine and TPA was expressed as %of control incubations containing no piperine or TPA. The experimentswere repeated using melanoblasts from 3 different donors.

Results

[0105]FIG. 6 shows the effect of piperine on the growth of humanmelanoblasts in vitro. Piperine at the concentrations of 1, 10, 100 μMwas found to cause significant stimulation to human melanoblasts in adose response manner, with 34% more cell yield compared to vehiclecontrol when the culture was exposed to 100 μM piperine in culture for 5days. TPA, a well-known melanocytic growth-stimulating agent, was alsoable to cause significant cell growth at tested concentrations, withover 50% of more cell yield observed when the culture was exposed to 20nM for 5 days. In the other repeated experiments, piperine wasconsistently observed to induce significant cell growth at theconcentrations ranging from 5-100 μM; these stimulatory effects weregenerally less than that of TPA. Morphologically, in the presence ofpiperine, melanoblasts appeared to be more dendritic and the cell bodieswere flatter and smaller.

Example 9

[0106] Experiments on human melanocytes in culture

[0107] Human melanocytes used in this experiment were derived frominduced differentiation of human foetal melanoblasts. The key characterof human melanocytes that is different from its precursor melanoblastsis their ability to synthesise melanin. Melanin is a valid marker formelanocytes. The cell pellet of human melanocytes exhibits acharacteristic brown to black colour, whereas human melanoblasts cannotproduce melanin thus devoid of brown or black colour in the cell pellet.

[0108] Two protocols were employed for the experiments on humanmelanocytes in culture. The first employed 24-well plates and evaluatedcell number with SRB assay. The second employed petri dishes and cellnumber was counted with a haemocytometer chamber.

[0109] For the first protocol, subconfluent human melanocytes maintainedin a Ø100 mm petri dish were subcultured into two 24-well plates(Falcon) using basic culture medium of RPMI 1640 supplemented with FBS(10%), bFGF (100 pM), CT (1 nM) and endothelin 1 (1 nM). The initialplating density was 20,000 cells/cm² (38,200 cells/well) with each wellcontaining 1000 μl medium. After incubation in a 10% CO₂, humidifiedatmosphere, at 37° C. for 2-3 hours to allow the attachment of thecells, piperine in 500 μl medium was added into wells to made up finalconcentrations of 0, 1, 5, 10 and 100 μM. Cells only in the medium withabove supplement lacking of endothelin I were also set up as negativecontrol. Six replicates were used in each group of treatment, andculture was incubated for 5 days before the cells were harvested byfixing with cold TCA (final concentration 20%) and processed with SRBassay. The solubilized SRB dye solution was transferred to a 96-wellplate for optical density reading.

[0110] For the second protocol, subconfluent human melanocytes weresubcultured in a Ø60 mm petri dishes (28 cm², Falcon) with RPMI 1640basic medium supplemented with FBS (10%), CT (1 nM), bFGF (100 pM) andendothelin 1 (1 nM). The initial plating density was 10,000 cells/cm²,with 5 ml medium per dish. Cells were incubated for 2-3 hours in 10%CO₂, humidified atmosphere, at 37° C., followed by addition of piperinesolution in to the dishes, making the final concentrations of 0, 1, 5,10 and 100 μM. Cells in the above supplemented medium lacking endothelin1 were also set up as a negative control. Three dishes were used foreach treatment and the culture was maintained for 5 days before cellswere harvested with trypsinisation and counted with a haemocytometerchamber. For melanin production experiment, the harvested cells werecentrifuged and pelleted. After carefully removing the medium, NaOH (1M) was used to solubilized the cell pellets and optical density read at475 nm in a Perkin-Elmer UV spectrophotometer (model UV/VIS Lambda 2).The melanin content was calculated by using a regression equationy=0.005+0.005x corresponding to the calibration curve for syntheticmelanin.

Results

[0111]FIG. 7 delineates the effects of piperine on the growth of humanmelanocytes cultured in 24-well plate. Piperine at the concentrations of5 and 10 μM markedly stimulates the growth of these pigmented cells,with 36% more cells yielded when the culture was under the influence of10 μM piperine for 5 days. However, at 100 μM, piperine exertedinhibitory effect on the growth of these cells. In addition, in thepresence of 1 nM endothelin 1, TPA at 20 nM was not able to stimulatecell growth in our culture system, a result that is of great differencewith that observed in human melanoblasts.

[0112] Table 5 shows the effects of piperine on the growth of humanmelanocytes cultured in petri dishes. It is conspicuous that in thepresence of ET1 (1 nM), piperine at the concentrations of 5 and 10 μMsignificantly stimulated the growth of human melanocytes, with cellnumber over twice as many as that of ET1 (1 nM) control when thismelanocyte cell type was exposed to 5 μM piperine for 5 days. Thisresult was consistent with that obtained from the 24-well plateexperiments, and it served to confirm that the stimulatory effectsobserved by SRB assay were indeed due to increased cell number ratherthan augmentation of protein production alone. TABLE 1 Preliminaryscreening of 30 herbal aqueous extracts on the proliferation of melan-acell line detected with SRB assay after 4 days culture. Cell number (%of control) after 4 days incubation when grown in the presence ofextract at: Names of herbs Plant part 1 mg ml⁻¹ 0.1 mg ml⁻¹ 0.01 mg ml⁻¹plants with a significant stimulatory effect Astragalus membranaceous(Fisch.) Bunge Root 163.2* 123.6* 105.6 Citrus reticulata Blanco (QingPi - unripe) Peel 16.0 138.5* 127.6* Dictamnus dasycarpus Turcz. rootbark 105.0 159.4* 98.0 Ophiopogon japonicus (Thunb.) Kergawe Root 127.8*126.5* 108.4 Piper nigrum L. Fruit 11.5 215.4* 151.3* Poria cocos(Schw.) Wolf (fungus) Sclerotium 79.0 134.6* 128.8* Tribulus terrestrisL. Fruit 80.7 136.1* 142.2* plants with no significant stimulatoryeffect Angelica dahurica (Fisch.) Benth. & Hook. Root 50.4 118.3 107.0Chaenomeles lagenaria (Loisel.) Koldz. fruit 57.1 74.5 99.0 Citrusreticulata Blanco (Chen Pi - ripe) Peel 34.6 101.1 81.1 Corydalisbulbosa D.C. Root 91.8 101.2 92.9 Curcuma longa L. Root 84.1 104.8 108.3Cyperus rotundus L. Rhizome 27.5 52.8 55.8 Cornus officinalis Sieb. etZucc. Fruit 30.4 92.1 101.6 Gentiana scabra Bunge Root 42.2 107.4 108.6Ligustrum lucidum Ait. Fruit 97.6 58.1 98.4 Lithospermum erythrorhizonSieb. et Zucc. Root 43.8 103.8 111.3 Notopterygium incisium Tingroot/rhizome 18.1 97.4 94.8 Paeonia lactiflora Pall. Root 31.8 62.2100.7 Paeonia suffruticosa Andr. Root 53.2 72.0 132.8 Picrorhiza kurroaRoyle ex. Benth Rhizome 42.5 77.5 90.0 Platycodon grandiflorum (Jacq.)A. DC. Root 35.1 94.1 96.8 Plumbago zeylanica L. Root 30.2 103.9 114.1Polygala tenuifolia Willd. Root 12.7 43.7 79.6 Ramulus mari (insect)Whole 41.1 87.1 89.4 Siesgesbeckia pubescens Makino Herb 17.0 40.8 51.7Spirodela polyrrhiza (L.) Scheid Herb 100 79.3 96.6 Trichosantheskirilowii Maxim Root 112.9 108.1 116.1 Tripterygium wilfordii Hook. Root89.8 36.7 63.3 Zingiber officinale Roscoe Rhizome 7.9 105.8 90.6

[0113] TABLE 2 Effects of Piper nigrum extract on the proliferation ofmelan-a cells counted with haemocytometer Treatment to cells cell number(×10⁻⁴/ml) Control 2.02 20 nM TPA 5.0* Piper nigrum at 0.01 mg/ml 3.06*Piper nigrum at 0.1 mg/ml 3.13*

[0114] TABLE 3 Biological origin and the culture conditions of a panelof different cell types used in selectivity experiment. optimum cultureconditions Cell name biological origin FBS Medium incubation (4 days)Melan-a normal epidermal melanoblasts from embryos of inbred  5%RPMI1640 37° C., 10% CO₂ C57BL mice Melan-c albino embryos of outbredLAC-MF strain mice 10% RPMI1640 37° C., 10% CO₂ FM21E human foetalmelanoblasts from epidermis (strain 21) 10% MCDB153 37° C., 10% CO₂melanoblast FM21E Human melanocytes derived from FM21E melanoblastsRPMI1640 37° C., 10% CO₂ melanocyte SVK14 human keratinocytes 10% DMEM37° C., 10% CO₂ CSM14.1.4 neuronal cells from mesencephalin of rat 10%DMEM 34° C., 5% CO₂ SC1 Fibroblastoids from neonatal murine skin 10%DMEM 37° C., 10% CO₂ XB2 murine keratinocytes 10% DMEM 37° C., 10% CO₂B6F10 mouse melanoma  5% RPMI1640 37° C., 10% CO₂ CACO2 human coloncancer 10% RPMI1640 37° C., 10% CO₂ IM9 human lymphoblastoid B cells 10%RPMI1640 37° C., 5% CO₂ Swiss 3T3 mouse fibroblasts 10% DMDM 37° C., 5%CO² Human healthy human blood samples 10% DMEM 37° C., 5% CO₂lymphocytes

[0115] TABLE 4 Effects of piperine and TPA on the growth of a panel ofcell types. (see Table 3 for details of cells) cell number as a % ofcontrol piperine at the TPA at the concentration of (μM) concentrationof Cell type 0.01 0.1 1 10 100 20 nM 200 nM Melan-a ND 130* 169* 153* ND295* ND Melan-c 109 208* 198* 119* 137* 186* 222* FM21E ND 101 101 119*134* 153* ND Melanoblast FM21E human ND ND  98 143*  75*  98 102melanocytes SVK14  97 101  92  84*  23*  71*  66* CSM14.1.4  93  94  95 89  64*  85*  76* SC1 191* 178* 175* 204* 190* 178* 199* XB2  80  90 86  90  42*  96  99 B16F10  71*  64*  47*  33*  0*  35*  55* CACO2 103 99 102  81*  34*  95  90 IM9 ND 101 103  69* ND ND ND Swiss 3T3 113 104106 102  51* 185* 207* Human Lymph- ND  93  93 ND ND 282* ND ocytes

[0116] TABLE 5 Effects of piperine on the proliferation and melaninproduction of human melanocytes cultured in petri dishes Cell no.(×10⁻⁴) ± SD after cultured for 5 % of control OD reading ± SD MelaninTreatments days (ET1 1 nM) at 475 nm content/10⁴ cells Cells only 17.71± 6.16* 49.1 0.026 ± 0.0049

 0.23 ± 0.001 μg ET1 (1 Nm) 36.04 ± 6.16  100.0  0.087 ± 0.044   0.46 ±0.01 μg ET1 (1 nM) + 60.83 ± 16.78 168.8 0.134 ± 0.014

  0.42 ± 0.03 μg piperine (1 μM) ET1 (1 nM) + 78.96 ± 5.63* 219.1 0.137± 0.0085

0.334 ± 0.01 μg  piperine (5 μM) ET1 (1 nM) +  64.79 ± 13.47* 179.80.139 ± 0.028

  0.41 ± 0.07 μg Piperine (10 μM) ET1 (1 nM) + 61.04 ± 10.04 169.4 0.144± 0.0046

 0.46 ± 0.001 μg piperine (100 μM)

Example 10 Derivatives of Piperine

[0117] 1.0 Introduction

[0118] Vitiligo is defined as a circumscribed, acquired, idiopathic,progressive hypomelanotic skin disorder which is characterised by thedevelopment of patchy depigmented macules due to progressive loss ofmelanocytes which is often familial with lack of established aetiology.

[0119] Various piperine derivatives of formula (1) were synthesised andtested for melanocyte (mouse melan-a) proliferant activity in-vitro.Cells were incubated with the text compound for 4 days, after which thesulphorhodamine-B (SRB) assay was performed to determine cell number SRBuptake was measured as optical density at 550 nm. The control assay wascarried out on cells incubated without test compound. There were 2 or 3series of experiments, each of which consisted of six replicateexperiments. The results are tabulated below.

[0120] 1.1 Percentage cell growth (A)

[0121] Percentage cell growth was obtained with a given compoundcalculated as (optical density in the presence of the compound/ controloptical density)×100.

[0122] 1.2 Relative activity to piperine

[0123] Melan-a cell proliferant activity for tested compounds wascompared with that obtained with piperine. Percentage stimulant activityis (A-100) where A stands for piperine or a test compound's percentagecell growth (see 1.1). All figures are given with Standard Error ofMeasurement.

[0124] Relative activity to piperine was calculated as (A-100)compound/(A-100) piperine).

[0125] Interpretation of the relative active value is as follows

[0126] <0—Inhibition of cell growth

[0127] 0—No effect (equal to control)

[0128] 0-1—Stimulant but weaker effect than piperine

[0129] 1—Equal stimulant effect to piperine

[0130] >1—Stimulant and stronger effect than piperine

[0131] 1.3 Dendricity

[0132] Effect on dendricity of melan-a cells by the test compounds wasby observation under microscope. Dendricity is relevant to vitiligosince normal skin melanocytes have dendrites, but in vitiligo themelanocytes seem to lose these before they disappear from the patches.

[0133] 1.4 Synthesis of piperine analogues

[0134] Analogues of piperine were synthesised using methods described inthe literature, adapted from the literature or devised in the inventors'laboratory. Structures of compounds were verified using NMR, MS, IRspectroscopy and melting point. Unless a synthetic method is given,reagents and reactants were purchased from Sigma Aldrich.

[0135] 1.5 Results

[0136] Table 6 presents an overall summary of the results appearing indetail in other Tables which follow. Tables 7-12 relate to results at asingle concentration of test compound (10 μM). They are followed by datashowing results at other concentrations. Many compounds showed a“cross-over” effect in which the test compound was less active thanpiperine at 10 μM but more active at 50 μM. This is illustrated for onecompound (RV-A01) in FIG. 8 of the Drawings. TABLE 6 Overall Summary ofResults More active Active at than piperine at Test Change with Respectto Active at higher conc. higher conc. CPD. Pipeline 10 μM? (μM)? (μM)?Vary amide (amino group listed below) RV-A01 Pyrrolidino Yes 25, 50 50RV-A02 Morpholino Yes 25, 50 50 RV-A04 3,4-methylenedioxy- Yes^(a) 50 50benzylamino RV-A05 Hexylamino Yes 25, 50 No RV-A06 Isobutylamino Yes 25No RV-A07 Methylamino Yes No No RV-A08 Ethylamino Yes No No RV-A09Isopropylamino Yes No No RV-A10 Cyclohexylamino Yes 50 50 RV-A11Butylamino Yes 50 50 Shorten connecting chain by 2 C-atoms (1 doublebond) RV-B01 Piperidino Yes 25, 50 50 Shorten connecting chain by 2C-atoms (1 double bond) and vary amino part of amide group RV-B02Pyrrolidino No^(b) Not done^(d) Not done RV-B03 Morpholino No Notdone^(d) Not done Replace amide by ester (alkyl group listed below)RV-AB1 Methyl Yes 50, 100 50, 100 RV-AB2 Ethyl No^(c) Not done Not doneRV-AB4 Isopropyl Yes 50 50 RV-AB5 Propyl Yes 50  50, 100^(e) RV-AB6Butyl Yes 50, 100 50, 100 Shorten connecting chain as above and replaceamide by ester (alkyl group listed below) RV-BB1 Methyl No^(b) Not doneNot done Reduce double bonds in connecting chain, making it saturatedRV-C02 Yes 25, 50 No Reduce double bonds in connecting chain and shortenit by 2 C-atoms RV-C03 No^(b) Not done Not done Replace3′,4′-methylenedioxy by methoxy and shorten connecting chain by 2C-atoms (1 double bond) RV-G01 6′-MeO No  100^(e) No RV-G02 3′-MeO No 100^(e) No RV-G03 4′-MeO No 100 No RV-G04 3′,4′-Di-MeO No 100 No

[0137] TABLE 7 Effect on melan-a cells at μM concentration Percentagecell growth Relative Variation on Nitrogen Substituent of Piperine(Repeated experiments) Stimulant activity to Den- Code N° Structure Testcpd. Piperine activity piperine dricity RV-A01

183 ± 34** 202 ± 84* 180 ± 50** 191 ± 63* Positive 1.03 1.01 +++ RV-A02

156 ± 58 187 ± 40** 153 ± 19** 210 ± 65** 170 ± 22 155 ± 19** Positive0.5 1.02 0.9  +++ RV-A04

149 ± 47 119 ± 27 147 ± 22 170 ± 39** 169 ± 29** 173 ± 28** Non-significant here, but positive in dose response test 0.7 0.27 0.66 +RV-A05

166 ± 35 140 ± 17* 170 ± 39** 169 ± 29** Positive 0.93 0.57 +++ RV-A06

147 ± 66 158 ± 24** 156 ± 40** 170 ± 39** 169 ± 29** 155 ± 18** Positive1.69 0.83 1.0  +++ RV-A07

170 ± 24* 216 ± 33* Positive 0.6  ++ RV-A08

200 ± 14 236 ± 17 Positive 0.73 +++ RV-A09

224 ± 19 263 ± 16** Positive 0.76 +++ RV-A10

308 ± 29** 302 ± 17** Positive 1.02 +++ RV-A11

264 ± 21** 347 ± 14** Positive 0.66 +++

[0138] TABLE 8 Effect on melan-a cells at μM concentration Variation inconnecting chain length and amide Percentage cell growth Relative group(Repeated experiments) Stimulant activity to Code N^(o) Structure TestPiperine activity piperine Dencdricity RV-B01

171 ± 33** 148 ± 20 152 ± 22** 180 ± 50** 191 ± 63** 155 ± 18** Positive0.88 0.52 0.97 ++ RV-B02

140 ± 14 154 ± 33 135 ± 4 180 ± 50** 191 ± 63** 155 ± 18** Non-significant 0.2 0.59 0.63 + RV-B03

103 ± 12 116 ± 17 210 ± 65** 170 ± 22** None 0.02 0.22 −

[0139] TABLE 9 Effect on melan-a cells at μM concentration Percentagecell growth Relative Replacement of amide by ester group (Repeatedexperiments) Stimulant activity to Code N^(o) Structure Test Piperineactivity piperine Dendricity RV-AB1

163 ± 38 141 ± 18* 151 ± 7* 210 ± 65** 170 ± 22** 155 ± 18** Positive0.57 0.59 0.93 ++ RV-AB2

29 ± 9 22 ± 0.4 171 ± 39** 171 ± 39** Positive −1 −1.09 Toxic RV-AB4

224 ± 12** 255 ± 15** Positive 0.8 ++ RV-AB5

166 ± 35** 169 ± 29** Positive 0.95 ++ RV-AB6

148 ± 18** 181 ± 11** Positive 0.59 +

[0140] TABLE 10 Effect on melan-a cells at μM concentration Replacementof amide by ester group and Percentage cell growth Relative variation inconnecting chain length (Repeated experiments) Stimulant activity toCode N^(o) Structure Test Piperine activity piperine Dendricity RV-BB1

149 ± 27 129 ± 15 121 ± 12 210 ± 65** 170 ± 22** 155 ± 18** Non-significant 0.44 0.41 0.39 +

[0141] TABLE 11 Effect on melan-a cells at μM concentration Reduction ofdouble bonds in connecting chain and variation in Percentage cell growthRelative chain length (Repeated experiments) Stimulant activity to CodeN^(o) Structure Test Piperine activity piperine Dendricity RV-C02

169 ± 29** 195 ± 89** 180 ± 50** 191 ± 63** Positive 0.8 1.04 +++ RV-C03

104 ± 5 113 ± 2 171 ± 7** 171 ± 7** None 0.056 0.18 + RV-C04

192 ± 5** 216 ± 18** Positive 0.79 +++ RV-C05

160 ± 5** 192 ± 2** Positive 0.65 ++

[0142] TABLE 12 Effect on melan-a cells at μM concentration Variation inthe phenyl substituent and Percentage cell growth Relative connectingchain length (Repeated experiments) Stimulant activity to Code N^(o)Structure Test Piperine activity piperine Dendricity RV-G01

105 ± 8 202 ± 29** None 0.04 RV-G02

119 ± 18 87 ± 17 171 ± 39** 171 ± 7** Negative 0.26 −0.18 − RV-G03

121 ± 8 122 ± 8* 171 ± 39** 171 ± 7** Non- significant 0.29 0.30 −RV-G04

100 ± 9 224 ± 11** None 0 —

[0143] Code N^(o) Structure RV-A01

Com- pounds Tested 1 μM 10 μM 25 μM 50 μM Piperine 151 ± 7**♦ 202 ±12**♦ 171 ± 15**♦ 142 ± 9** RV- 109 ± 7 122 ± 7 142 ± 21** 186 ± 14**A01

[0144] Code N^(o) Structure RV-A02

Com- pounds Tested 1 μM 10 μM 25 μM 50 μM Piperine 147 ± 192 ± 13**♦ 167± 19** 142 ± 15** 11**♦ RV- 125 ± 10 167 ± 17** 171 ± 8** 168 ± 12** A02

[0145] Code N^(o) Structure RV-A04

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 120 ± 11 178 ± 11**♦116 ± 13 92 ± 9 RV-A04 101 ± 12 138 ± 10** 150 ± 15** 71 ± 9 Dentricity− + + RV-A04

[0146] Code N^(o) Structure RV-A05

Com- pounds Tested 1 μM 10 μM 25 μM 50 μM Piperine 173 ± 6**♦ 230 ±13**♦ 188 ± 19** 182 ± 15** RV- 155 ± 9** 188 ± 13** 178 ± 18** 174 ±8** A05

[0147] Code N^(o) Structure RV- A06

Compounds Tested 1 μM 10 μM 25 μM 50 μM Piperine 147 ± 8♦ 195 ± 22** 173± 17* 159 ± 14 RV-A06 134 ± 7 188 ± 14** 172 ± 15* 135 ± 24

[0148] Code N^(o) Structure RV-A07

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 211 ± 16**♦ 216 ± 33**52 ± 15 16 ± 3 RV-A07 140 ± 12** 170 ± 24** 71 ± 5 46 ± 2 Dentricity ++++ + + of RV-A07

[0149] Code N^(o) Structure RV-A08

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 216 ± 14**♦ 236 ± 17**61 ± 11 32 ± 5 RV-A08 139 ± 27** 200 ± 14** 81 ± 12 62 ± 13 Dendricity++ +++ + + of RV-A08

[0150] Code N^(o) Structure RV-A09

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 221 ± 17**♦ 263 ± 16**77 ± 12 24 ± 2 RV-A09 187 ± 15** 224 ± 19** 85 ± 5 42 ± 6 Dendricity ++++++ + + of RV-A09

[0151] Code N^(o) Structure RV- A10

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 236 ± 30** 302 ± 17**78 ± 11 21 ± 4 RV-A10 301 ± 20** 308 ± 29** 155 ± 22** 100 ± 13Dendricity +++ +++ ++ + of RV-A10

[0152] Code N^(o) Structure RV-A11

Com- pounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 251 ± 19**♦ 347 ±14**♦ 61 ± 7 25 ± 2 RV-A11 189 ± 6** 264 ± 21** 158 ± 20** 84 ± 6Dendricity +++ +++ ++ + of RV-A11

[0153] Code N^(o) Structure RV-B01

Compounds Tested 1 μM 10 μM 25 μM 50 μM Piperine 144 ± 27**♦ 190 ± 7**172 ± 11** 153 ± 10** RV-B01 111 ± 6 147 ± 7** 187 ± 18** 187 ± 8**

[0154] Code N^(o) Structure RV-AB1

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 133 ± 31** 177 ± 14**♦139 ± 16* 95 ± 24 RV-AB1 125 ± 13 147 ± 16** 187 ± 12** 171 ± 8**Dendricity − + ++ ++ of RV-AB1

[0155] Code N^(o) Structure RV-AB4

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 223 ± 18**♦ 255 ± 15**60 ± 16 24 ± 6 RV-AB4 175 ± 6** 224 ± 12** 148 ± 19** 90 ± 7 Dendricity++ ++ ++ + of RV-AB4

[0156] Code N^(o) Structure RV-AB5

Com- pounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 141 ± 26**♦ 220 ±29**♦ 45 ± 12 23 ± 4 RV-AB5 120 ± 21 151 ± 19** 163 ± 8** 123 ± 8Dendricity − ++ ++ + of RV-AB5

[0157] Code N^(o) Structure RV-AB6

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 113 ± 10 181 ± 11** 43± 6 23 ± 6 RV-AB6 103 ± 5 148 ± 18** 190 ± 11** 128 ± 17** Dendricity− + ++ + of RV-AB6

[0158] Code N^(o) Structure RV-C02

Compounds Tested 1 μM 10 μM 25 μM 50 μM Piperine 158 ± 203 ± 11** 188 ±12** 164 ± 6** 10**♦ RV-C02 134 ± 15** 183 ± 33** 199 ± 31** 175 ± 12**

[0159] RV-C04 Code N^(o) Structure RV-C04

Com- pound 1 μM 10 μM 50 μM 100 μM Piperine 191 ± 12**♦ 216 ± 18** 184 ±6** 96 ± 6 RV-C04 129 ± 6** 192 ± 6** 192 ± 10** 191 ± 12** Dendricity ++++ +++ +++ of RV-C04

[0160] RV-C05 Code N^(o) Structure RV-C05

Compound 1 μM 10 μM 50 μM 100 μM Piperine 161 ± 13** 192 ± 2**♦ 189 ±15** 87 ± 13 RV-C05 118 ± 1 160 ± 5**♦ 158 ± 19** 113 ± 15 Dendricityof + ++ ++ + RV-C05

[0161] Code N^(o) Structure RV-G01

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 161 ± 23** 202 ± 29**61 ± 5 40 ± 7 RV-G01 99 ± 8 105 ± 8 103 ± 6 119 ± 9 Dendricity − − − −of RV-G01

[0162] Code N^(o) Structure RV-G02

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 151 ± 17** 201 ± 15**57 ± 15 39 ± 11 RV-G02 99 ± 5 95 ± 18 110 ± 11 127 ± 9 Dendricity − − −− of RV-G02

[0163] Code N^(o) Structure RV-G03

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 163 ± 9** 181 ± 23**59 ± 11 40 ± 12 RV-G03 90 ± 10 108 ± 20 111 ± 10 133 ± 15** Dendricity −− − − of RV-G03

[0164] Code N^(o) Structure RV-G04

Compounds Tested 1 μM 10 μM 50 μM 100 μM Piperine 179 ± 12** 224 ± 11**92 ± 19 35 ± 4 RV-G04 95 ± 11 100 ± 9 114 ± 8 123 ± 7* Dendricity − − −− of RV-G04

[0165] 2. Synthesis of amide derivatives of piperinic acid

[0166] 2.1 Preparation of piperinic acid (RV-A00)

[0167] To piperine (1) (2 g, 0.7 mmol, 1 eq), 20% of methanolic KOH (100ml) was added and refluxed for 2 days. After completion of thehydrolysis, methanol was removed under reduced pressure and a yellowcoloured oily solid was obtained. This residue was dissolved in water(50 ml) and acidified with 6N HCl to pH<1 yielding a yellowishprecipitate of piperinic acid. Recrystallization from methanol gaveyellow needles (0.9 g, 60% yield). m.p. 206°-208° C. (Lit m.p. 217°-218°C.)¹

[0168] 2.2 Synthesis of piperlonguminine (RV-A06)

[0169] A mixture of piperinic acid (350 mg, 0.0016 mole, 1 eq) andtriethylamine (0.4 ml, 0.0032 mole, 2 eq) in dichloromethane (50 ml) wasstirred for 15 min at 0° C. To this mixture methanesulfonyl chloride(0.18 ml, 0.0024 mole, 1.5 eq) was added and stirred for further 30 minat 0° C. Isobutylamine (0.23 ml, 0.0024 mole, 1.5 eq) was added to themixture and stirred for 1 h at 0° C. and 2 h at room temperature.Dichloromethane (50 ml) was added to the mixture which was then washedwith 5% HCl (3×100 ml), saturated aqueous NaHCO₃ (3×100 ml) and water(3×100 ml). The organic fraction was dried over anhydrous sodiumsulphate, filtered and rotary evaporated to yield a yellowish solidresidue. Recrystallisation from methanol yielded colourless needles ofpiperlonguminine (120 mg, 32% yield)². The reaction is presumed toproceed through a mesylate ester intermediate.

[0170] Piperlonguminine (RV-A06)

[0171]¹H-NMR (CDCl₃) δ: 5.96 (d, 1H, J=14.8, CH═CH—CH═CH), 7.36 (d,d,1H, J=10.5, 14.8, CH═CH—CH═CH), 6.66 (d,d, 1H, J=15.4, 10.5,CH═CH—CH═CH), 6.76 (d, 1H, J=15.4 CH═CH—CH═CH), 6.96 (d,1H J=1.6,Ar-7H), 6.76 (d,1H J=8.0, Ar-10H), 6.87 (d, d, 1H J=1.6, 8.0 Ar-11H),5.97 (s, 2H, O—CH₂—O), 3.18 (t, 2H, J=6.5 CH₂—CH), 1.83 (m, 1H, J=6.5CH₂—CH), 0.94(d, 6H, J=6.5, (CH₃) ₂), 5.82 (t, 1H, NH J=5.3)

[0172]¹³C-NMR (CDCl₃): 20.4 (CH₃), 29.4 (CH), 47.3 (CH₂), 102.2 (CH₂),106.2 (CH), 109.1 (CH), 123.3(CH), 125.5 (CH), 126.0 (CH), 132.0 (C),138.0 (CH), 140.4 (CH), 148.9 (C), 149.2 (C), 166.2 (C)

[0173] MS m/z (%): 273 (M⁺ 98), 216 (20), 201 (100), 174 (25), 173 (65),172 (23), 171 (17) 143 (20), 115 (40), 96 (11).

[0174] IR (kBr): v_(max) (carbonyl group)1644

[0175] m.p. 161.2°-161.7° C. (Lit m.p. 156°-160° C.) ¹

[0176] 2.3 Synthesis of other amide derivatives of piperinic acid

[0177] The general method was as for piperlonguminine (section 1.2),using the same proportions of reactive amine, triethylamine andmethanesulfonyl chloride relative to piperinic acid (200 or 300 mg., 1eq). Recrystallisation from ethyl acetate/petroleum spirit yielded theother amide derivatives of piperinic acid.

[0178] 5-E,E-piperinoylpyrrolidine (RV-A01)

[0179]¹H-NMR (CDCl₃) δ: 6.26 (d, 1H, J=14.7, CH═CH—CH═CH), 7.43 (d,d,1H, J=9.5, 14.7, CH═CH—CH═CH), 6.73 (d,d, 1H, J=15.3, 9.5, CH═CH—CH═CH),6.78 (d, 1H, J=15.3 CH═CH—CH═CH), 6.98 (d,1H J=1.6, Ar-7-H), 6.77 (d,1HJ=8.0, Ar-10-H), 6.89 (d, d, 1H J=1.6, 8.0 Ar-11-H), 5.97 (s, 2H,O—CH₂—O), 3.57 (t, 2H, J=4.0 N—CH₂ (pyrrolidine)) 3.54 (t, 2H, J=4.0N—CH₂ (pyrrolidine) 1.90 (m, 2H, CH₂CH₂(pyrrolidine)) 1.87 (m, 2H,CH²⁻CH₂(pyrrolidine))

[0180]¹³C-NMR (CDCl₃): 24.3 (CH₂), 26.1 (CH₂), 45.9 (CH₂), 46.4 (CH₂),101.2 (CH₂), 105.7 (CH), 108.4 (CH), 121.4 (CH), 122.5 (CH), 125.2 (CH),130.9 (C), 138.7 (CH), 141.7 (CH), 148.1 (C), 148.2 (C), 164.9 (C)

[0181] MS m/z (%): 271 (M⁺ 78), 201 (100), 173 (30), 172 (15), 171 (13)143 (13), 115 (27)

[0182] IR (KBr): v_(max) (carbonyl group) 1637

[0183] m.p. 142.9°-143° C. (Lit m.p. 142°-143° C.)², yield 49.2%

[0184] 5-E,E-piperinoyl morpholine (RV-A02)

[0185]¹H-NMR (CDCl₃) δ: 6.37 (d, 1H, J=14.6, CH═CH—CH═CH), 7.45 (d,d,1H, J=10.2, 14.6, CH═CH—CH═CH), 6.72 (d,d, 1H, J=15.5, 10.2,CH═CH—CH═CH), 6.79 (d, 1H, J=15.5 CH═CH—CH═CH), 6.98 (d,1H J=1.5,Ar-7-H), 6.80 (d,1H J=8.0, Ar-10-H), 6.89 (d, d, 1H J=1.5, 8.0 Ar-11-H),5.98 (s, 2H, O—CH₂—O), 3.70 (t, 2H, J=4.0 CH₂—N—CH₂ (morpholine)) 3.60(t, 2H, J=4.0 CH₂—O—CH₂ (morpholine))

[0186]¹³C-NMR (CDCl₃): 42.3 (CH₂), 46.1(CH₂), 66(CH₂), 66(CH₂), 101.3(CH₂), 106.5 (CH), 108.5 (CH), 118.7 (CH), 122.7 (CH), 124.9 (CH), 130.8(C), 139.1 (CH), 143.4 (CH), 148.2 (C), 148.3 (C), 165.6 (C)

[0187] MS m/z (%): 287 (M⁺ 57), 201 (100), 173 (25), 171 (10) 143 (10),115 (30)

[0188] IR (KBr): v_(max) (carbonyl group) 1641

[0189] m.p. 161.8°-162.5° C. (Lit m.p. 167-168° C.)³, yield 44.1%

[0190] 5-E,E-piperinoylpiperinolymine (RV-A04)

[0191]¹H-NMR (CDCl₃) δ: 5.98(d, 1H, J=14.9, CH═CH—CH═CH), 7.34 (d,d, 1H,J=10.7, 14.9, CH═CH—CH═CH), 6.73 (d,d, 1H, J=15.5, 10.7, CH═CH—CH═CH),6.79 (d, 1H, J=15.5 CH═CH—CH═CH), 6.98 (d,2H J=1.5, Ar-7,3′-H), 6.78(d,2H J=8.0, Ar-10,6′-H), 6.89 (d, d, 2H J=1.6, 8.0 Ar-11,7′-H), 5.98(s, 2H, O—CH₂—O), 5.93 (s, 2H, O—CH₂—O), 4.40 (d, 2H, CH₂) 3.57 (br, 1H,NH)

[0192]¹³C-NMR (CDCl₃): 43.4 (CH₂), 101.1 (CH₂), 101.4 (CH₂), 105.8 (CH),108.3(CH) 108.5 (CH), 108.6 (CH), 121.2 (CH), 122.8 (CH), 124.7 (CH),130.9 (C), 132.2 (C) 139.9 (CH), 141.6 (CH), 147.0 (C) 147.9 (C)148.3(C), 148.4(C), 166.9 (C)

[0193] MS m/z (%): 351 (M⁺81), 216 (15), 203 (12), 202 (53) 201 (29),174 (31), 173 (22), 150 (23) 144 (11), 143 (10), 135 (100), 116 (12)115(29)

[0194] m.p. 190.5°-191.7° C., yield 50.1%

[0195] 5-E,E-piperinoylhexylamine RV-A05

[0196]¹H-NMR (CDCl₃) δ: 5.90 (d, 1H, J=14.8, CH═CH—CH═CH), 7.35 (d,d,1H, J=10.6, 14.8, CH═CH—CH═CH), 6.66 (d,d, 1H, J=15.4, 10.6,CH═CH—CH═CH), 6.76 (d, 1H, J=15.4 CH═CH—CH═CH), 6.97 (d,1H J=1.4,Ar-7H), 6.77 (d,1H J=8.0, Ar-10H), 6.88 (d, d, 1H J=1.5, 8.0 Ar-11H),5.97 (s, 2H, O—CH₂—O), 3.34 (q, 2H, CH₂—CH₂—CH₂—CH₂—CH₂) 1.54 (m, 2H,CH₂—CH₂—CH₂—CH₂—CH₂) 1.32 (m, 6H, CH₂—CH₂—CH₂—CH₂—CH₂) 0.88 (t, 3H,CH₃), 5.54 (br, NH)

[0197]¹³C-NMR (CDCl₃): 14.3 (CH₃), 22.5 (CH₂), 26.6 (CH₂), 29.6 (CH₂),31.5 (CH₂), 39.7 (CH₂), 101.3 (CH₂), 105.7 (CH), 108.5 (CH), 122.5 (CH),123.2 (CH), 124.6 (CH), 130.8 (C), 138.7 (CH), 140.9 (CH) 148.2 (C),148.2 (C), 166.0 (C)

[0198] MS m/z (%): 301(M⁺94), 202 (18) 201 (73), 174 (40), 173 (100),172 (31), 171 (15) 143 (24), 115 (63)

[0199] IR (KBr): v_(max) (carbonyl group)1641

[0200] m.p. 149.5°-149.8° C. (Lit m.p. 139°-141° C.)⁴, yield 50.1%

[0201] 5-E,E-piperinoylmethylamine (RV-A07)

[0202]¹H-NMR (CDCl₃) δ: 5.91 (d, 1H, J=14.8, CH═CH—CH═CH), 7.36 (d,d,1H, J=10.7, 14.8, CH═CH—CH═CH), 6.66 (d,d, 1H, J=15.4, 10.6,CH═CH—CH═CH), 6.77 (d, 1H, J=15.4 CH═CH—CH═CH), 6.97 (d,1H J=1.5,Ar-7H), 6.77 (d,1H J=8.0, Ar-10 H), 6.88 (d, d, 1H J=1.6, 8.0 Ar-11H),5.97 (s, 2H, O—CH₂—O), 2.91(t, 3H, CH₃), 5.61 (br, NH)

[0203]¹³C-NMR (CDCl₃): 26.9 (CH₃), 101.7 (CH₂), 106.1 (CH), 108.9 (CH),123.0 (CH), 123.3 (CH), 125.0 (CH), 131.2 (C), 139.2 (CH), 141.4 (CH),148.6 (C), 148.6 (C), 167.2 (C)

[0204] MS m/z (%): 231(M+89), 201 (42), 173 (67), 172 (32), 171 (17),143 (27), 116 (21) 115 (100), 89 (12)

[0205] m.p. 181.1°-182.4° C. (Lit m.p. 186° C.)⁵, yield 48.2%

[0206] 5-E,E-piperinoylethylamine (RV-A08)

[0207]¹H-NMR (CD₃OD) δ: 6.14 (d, 1H, J=15.0, CH═CH—CH═CH), 7.37 (d,d,1H, J=10.2, 15.0, CH═CH—CH═CH), 6.93 (d,d, 1H, J=15.7, 10.6,CH═CH—CH═CH), 6.87 (d, 1H, J=15.7 CH═CH—CH═CH), 6.97 (d,1H J=1.5,Ar-7H), 6.77 (d,1H J=8.0, Ar-10H), 6.88 (d, d, 1H J=1.6, 8.0 Ar-11H),5.97 (s, 2H, O—CH₂—O), 3.39 (m, 2H, J=6.2, CH₂),1.22(t, 3H, J=6.1, CH₃),

[0208]¹³C-NMR (CDCl₃): 14.7 (CH₃), 36.9 (CH₂), 103.2 (CH₂), 107.2 (CH),109.8 (CH), 121.2 (CH), 124.9 (CH), 125.9 (CH), 132.4 (C), 142.9 (CH),145.2 (CH), 150.2 (C), 150.6 (C),170 (C)

[0209] MS m/z (%): 245(M⁺ 78), 218 (34), 201 (71), 200 (49), 174 (64),173 (80), 172 (76), 171 (65), 143 (75), 116 (68),115 (100)

[0210] m.p. 158.5°-159.9° C. (Lit m.p. 162°-164° C.)⁴, yield 45.6%

[0211] 5-E,E-piperinoylisopropylamine (RV-A09)

[0212]¹H-NMR (CDCl₃) δ: 5.87 (d, 1H, J=14.8, CH═CH—CH═CH), 7.36 (d,d,1H, J=10.7, 14.8, CH═CH—CH═CH), 6.66 (d,d, 1H, J=15.4, 10.6,CH═CH—CH═CH), 6.76 (d, 1H, J=15.2 CH═CH—CH═CH), 6.97 (d,1H J=1.6,Ar-7H), 6.77 (d,1H J=8.0, Ar-10H), 6.88 (d,d, 1H J=1.6, 8.0 Ar-11H),5.97 (s, 2H, O—CH₂—O), 4.15(m, 1H, J=6.6, CH), 5.36 (d, 1H, J=7.3 NH),1.19 (d, 6H, J=6.6, (CH₃)₂)

[0213]¹³C-NMR (CDCl₃): 23.2 (CH₃)₂, 41.9 (CH), 101.9 (CH₂), 106.4 (CH),108.9 (CH), 123.0 (CH), 123.8 (CH), 124.1 (CH), 131.3 (C), 140.2 (CH),141.2 (CH), 148.8 (C), 148.6 (C) 165.6 (C)

[0214] MS m/z (%): 259(M⁺ 80), 201 (62), 174 (34), 173 (74), 172 (31),171 (15), 143 (30), 116 (16), 115 (100)

[0215] m.p. 169°-169.4° C. (Lit m.p. 171°-173° C.)⁴, yield 52%

[0216] 5-E,E-piperinoyl cyclohexylamine (RV-A10)

[0217]¹H-NMR (CDCl₃) δ: 5.93 (d, 1H, J=14.8, CH═CH—CH═CH), 7.35 (d,d,1H, J=10.6, 14.8, CH═CH—CH═CH), 6.66 (d,d, 1H, J=15.3, 10.6,CH═CH—CH═CH), 6.76 (d, 1H, J=15.4 CH═CH—CH═CH), 6.96 (d,1H J=1.6,Ar-7H), 6.76 (d,1H J=8.0, Ar-10H), 6.87 (d, d, 1H J=1.6, 8.0 Ar-11H),5.97 (s, 2H, O—CH₂—O), 3.87 (m, 1H, CH (cyclohexyl)) 1.99 (m, 2H,CH₂(cyclohexyl)) 1.65 (m, 4H, CH₂—CH₂(cyclohexyl) 1.39 (m, 2H,CH₂(cyclohexyl)) 1.18 (m, 2H, CH₂(cyclohexyl)) 5.48 (d,J=8.0 NH)

[0218]¹³C-NMR (CDCl₃): 25.3 ((CH₂)₂), 25.9 (CH₂), 33.6 ((CH₂)₂), 48.6(CH), 101.3 (CH₂), 101.7 (CH), 106.1 (CH), 108.9 (CH), 123.0 (CH), 124.0(CH), 125.1 (CH),131.3 (C), 139.0 (CH), 141.2 (CH) 148.5 (C), 148.5 (C),165.5 (C)

[0219] MS m/z (%): 299(M⁺56), 259 (48) 216 (33), 201 (60),174 (33), 173(61), 172 (18), 171 (16) 143 (17), 115 (100)

[0220] m.p. 196.4°-197.3° C. (Lit m.p. 199°-200° C.)⁴, yield 57.4%

[0221] 5-E,E-piperinoylbutylamine (RV-A11)

[0222]¹H-NMR (CDCl₃) δ: 5.97 (d, 1H, J=14.8, CH═CH—CH═CH), 7.35 (d,d,1H, J=10.7, 14.8, CH═CH—CH═CH), 6.66 (d,d, 1H, J=15.4, 10.6,CH═CH—CH═CH), 6.76 (d, 1H, J=15.4 CH═CH—CH═CH), 6.97 (d,1H J=1.6,Ar-7H), 6.77 (d,1H J=8.0, Ar-10H), 6.89 (d, d, 1H J=1.5, 8.0 Ar-11H),5.97 (s, 2H, O—CH₂—O), 3.36 (q, 2H, CH₂—CH₂—CH₂—) 1.54 (m, 2H,CH₂—CH₂—CH₂) 1.39 (m, 6H, CH₂—CH₂—CH₂) 0.93 (t, 3H, CH₃), 5.47 (br, NH)

[0223]¹³C-NMR (CDCl₃): 14.2 (CH₃), 20.5 (CH₂), 32.2 (CH₂), 39.8 (CH₂),101.7 (CH₂), 106.1 (CH), 108.9 (CH), 123.0 (CH), 123.6 (CH), 125.0 (CH),131.3 (C), 139.2 (CH), 141.3 (CH) 148.6 (C), 148.6 (C), 166.4 (C)

[0224] m.p. 144.2°-145.6° C. (Lit m.p. 144°-145° C.)⁴, yield 38.4%

References

[0225]¹Chatterjee, A., and Dutta, C. P. (1967). Alkaloids of Piperlongum Linn-I Structure and synthesis of piperlongumine andpiperlonguminine, Tetrahedron, 23,1769-1781.

[0226]²Nokio Nakumara, Fumiyuki Kiuchi, and Yoshisuke Tsuda (1988).Infrared spectra of conjugated amides: Reassignment of the C═O and C═Cabsorptions: Chemical and Pharmaceutical Bulletin, 36, 2647-2651.

[0227]³H. Oediger and A. Schulze (Bayer AG), (1979), DeutscheAuslegeschrift 2757 483

[0228]⁴Paula, Vanderlucia F. de; A Barbosa, Luiz C. de; Demuner, AntonioJ.; Pilo-Veloso, Dorila; Picanco, Marcelo C. (2000) Pest ManagementScience 56, 2, 168-174.

[0229]⁵Gokale et al., (1948) Journal of University Bombay Science 16/5A32-35

[0230] 3. Synthesis of ester derivatives of piperinic acid

[0231] 3.1 Preparation of piperinic acid (RV-A00)

[0232] As described above.

[0233] 3.2 Synthesis of 5-E,E-piperinic acid methyl ester (RV-AB1)

[0234] A mixture of piperinic acid (300 mg, 0.0014 mole, 1 eq) andtriethylamine (0.39 ml, 0.0028 mole, 2 eq) in dichloromethane (50 ml)was stirred for 15 min at 0° C. To this mixture methanesulfonyl chloride(0.16 ml, 0.0021 mole, 1.5 eq) was added and stirred for further 30 minat 0° C. Methanol in excess (10 ml) was added to the mixture and stirredfor 1 h at 0° C. and 1 h at room temperature. Dichloromethane (50 ml)was added to the mixture which was then washed with water (3×100 ml), 5%NaHCO₃ (3×100 ml) and water (3×100 ml). The organic fraction was driedover anhydrous sodium sulphate, filtered and rotary evaporated to yielda yellowish solid residue. Recrystallisation from ethylacetate/petroleum spirit yielded ester (180 mg, 56.2% yield). m.p.142.9°-143° C. (Lit m.p. 140° C.)⁶

[0235] 3.3 Synthesis of other esters of piperinic acid.

[0236] They were synthesised as described in section 3.2, replacingmethanol (10 ml) ethanol (10 ml), isopropanol, butanol or propanol (15ml).

[0237] 5-E,E-piperinic acid methyl ester (RV-AB1)

[0238]¹H-NMR (CDCl₃) δ: 5.94 (d, 1H, J=15.2, CH═CH—CH═CH), 7.41 (d,d,1H, J=10.8, 15.2, CH═CH—CH═CH), 6.70 (d,d, 1H, J=15.4, 10.8,CH═CH—CH═CH), 6.81 (d, 1H, J=15.7 CH═CH—CH═CH), 6.99 (d,1H J=1.6,Ar-7H), 6.79 (d,1H J=8.1, Ar-10H), 6.91 (d, d, 1H J=1.5, 8.1 Ar-11H),5.98 (s, 2H, O—CH₂—O), 3.57 (t, 3H, br, OCH₃J=4.7)

[0239]¹³C-NMR (CDCl₃) δ: 51.5(CH₃), 101.8 (CH₂), 106.2 (CH) 108.9 (CH),120.0 (CH), 123.4 (CH) 124.7 (CH), 130.8 (CH), 140.9 (C), 145.5 (CH),148.6 (C), 148.9 (C), 168.9 (C)

[0240] MS m/z (%): 232 (M⁺ 69), 201 (19), 174 (12), 173 (100), 172 (39),171 (12) 143 (33), 116 (11), 115 (53) 101 (15), 100(12)

[0241] 5-E,E-piperinic acid ethyl ester (RV-AB2)

[0242]¹H-NMR (CDCl₃) δ: 5.94 (d, 1H, J=15.2, CH═CH—CH═CH), 7.41 (d,d,1H, J=10.8, 15.3, CH═CH—CH═CH), 6.70 (d,d, 1H, J=15.4, 10.8,CH═CH—CH═CH), 6.81 (d, 1H, J=15.5 CH═CH—CH═CH), 6.99 (d,1H J=1.6,Ar-7H), 6.78 (d,1H J=8.1, Ar-10H), 6.91 (d, d, 1H J=1.6, 8.1 Ar-11H),5.98 (s, 2H, O—CH₂—O), 4.22 (q, 2H, OCH₂J=7.2), 1.31 (t, 3H, CH₃ J=7.2)

[0243]¹³C-NMR (CDCl₃): 14.7(CH₃), 60.7(CH₂), 101.6 (CH₂), 106.3 (CH)108.9 (CH), 120.8 (CH), 123.3 (CH)124.9 (CH), 131.0 (CH), 140.5 (CH),145.1 (CH), 148.7 (C), 148.9 (C), 167.6 (C)

[0244] 5-E,E-piperinic acid isopropyl ester (RV-AB4)

[0245] Physical data are not available for this compound.

[0246] 5-E,E-piperinic acid propyl ester (RV-AB5)

[0247]¹H-NMR (CDCl₃) δ: 5.94 (d, 1H, J=15.2, CH═CH—CH═CH), 7.41 (d,d,1H, J=10.7, 15.2, CH═CH—CH═CH), 6.70 (d,d, 1H, J=15.4, 10.8,CH═CH—CH═CH), 6.76 (d, 1H, J=15.4 CH═CH—CH═CH), 6.99 (d,1H J=1.6,Ar-7H), 6.78 (d,1H J=8.1, Ar-10H), 6.91 (d, d, 1H J=1.5, 8.0 Ar-11H),5.98 (s, 2H, O—CH₂—O), 4.12 (t, 2H, OCH₂ J=6.7) 1.69 (m, 2H, CH₂ J=7.3)0.97 (t, 3H, CH₃ J=7.4)

[0248]¹³C-NMR (CDCl₃): 10.9 (CH₃), 22.5 (CH₂), 66.3 (CH₂),101.8 (CH₂),106.2 (CH) 108.9 (CH), 120.9 (CH), 123.3 (CH)124.9 (CH), 131.0 (CH),140.5 (CH), 145.1 (CH), 148.7 (C), 148.9(C), 167.7 (C)

[0249] MS m/z (%): 260 (M⁺ 59), 201 (26), 174 (18), 173 (100), 172 (39),171 (14) 143 (34), 116 (16), 115 (73),100 (12)

[0250] m.p. 119°-120° C.

References

[0251]⁶Avijit Banerjee, Tapasree Ghosal, and Aditi Kacharya. (1984).Indian Journal of Chemistry, 23B, 546-549.

[0252] 5-E,E-piperinic acid butyl ester (RV-AB6)

[0253]¹H-NMR (CDCl₃) δ: 5.94 (d, 1H, J=15.2, CH═CH—CH═CH), 7.40 (d,d,1H, J=10.7, 15.3, CH═CH—CH═CH), 6.70 (d,d, 1H, J=15.4, 10.8,CH═CH—CH═CH), 6.76 (d, 1H, J=15.4 CH═CH—CH═CH), 6.99 (d,1H J=1.6,Ar-7H), 6.78 (d,1H J=8.0, Ar-10H), 6.91 (d, d, 1H J=1.5, 8.0 Ar-11H),5.98 (s, 2H, O—CH₂—O), 4.12 (t, 2H, OCH₂J=6.7) 1.69 (m, 2H, CH₂ J=7.3)1.69 (m, 2H, CH₂J=7.6), 0.95 (t, 3H, CH₃ J=7.5)

[0254] MS m/z (%): 274 (M⁺ 50), 201 (15), 174 (14), 173 (100), 172 (30),171 (14) 143 (21), 115 (55)

[0255] Obtained as an oil.

[0256] 4. Synthesis of amide derivatives of 3,4-methylenedioxycinnamicacid

[0257] These 3,4-methylenedioxycinnamide derivatives were synthesised asdescribed in Section 2.2, but using 3,4-methylenedioxycinnamic acid (500mg) as the starting acid and reducing the proportion of triethylamine to1.5 equivalent with respect to the starting acid. Also, in the firststage, the reaction mixture was stirred for 2 hours, instead of 30minutes, again at 0° C.

[0258] 1-(3-trans-benzo-1,3-dioxol-5-ylacryloyl)piperidine (RV-B01)

[0259]¹H-NMR (CDCl₃) δ: 7.56 (d, 1H, J=15.3, CH═CH), 6.73 (d, 1H,J=15.3, CH═CH—), 7.03 (d,1H J=1.5, Ar-7H), 6.79 (d,1H J=8.0, Ar-8H),6.99 (d, d, 1H J=1.6, 8.0 Ar-9H), 5.98 (s, 2H, O—CH₂—O), 3.57 (br, 2H,CH₂—N—CH₂), 3.65 (br, 2H, CH₂—N—CH₂(piperidine)), 1.65 (m, 6H,CH₂—CH₂—CH₂—(piperidine))

[0260]¹³C-NMR (CDCl₃): 24.8 (CH₂), 25.6 (CH₂), 26.7 (CH₂), 43.3 (CH₂),46.9 (CH₂), 101.3 (CH₂), 106.7 (CH), 108.4 (CH), 115.6 (CH), 123.5 (CH),129.9 (C), 141.9 (CH) 148.1 (CH), 148.8 (C), 165.4 (C)

[0261] m.p. 80.1°-82° C. (Lit m.p. 80°-82° C.)⁷, yield 49.2%

[0262] 1-(3-trans-benzo-1,3-dioxol-5-ylacryloyl)pyrrolidine (RV-B02)

[0263]¹H-NMR (CDCl₃) δ: 7.60 (d, 1H, J=15.2, CH═CH), 6.73 (d, 1H,J=15.3, CH═CH—), 7.04 (d,1H J=1.5, Ar-7H), 6.80 (d,1H J=8.0, Ar-8H),7.01 (d, d, 1H J=1.5, 8.0 Ar-9H), 5.99 (s, 2H, O—CH₂—O), 3.61 (br, 2H,CH₂—N—CH₂ (pyrrolidine)), 3.57 (br, 2H, CH₂—N—CH₂(pyrrolidine)), 1.99(4H, CH₂—CH₂(pyrrolidine)),

[0264]¹³C-NMR (CDCl₃): 24.3 (CH₂), 26.1 (CH₂) 46.0 (CH₂), 46.5 (CH₂),101.4 (CH₂), 106.4 (CH), 108.5 (CH), 116.8 (CH), 123.8 (CH), 129.7 (C),141.0 (CH) 148.1 (C), 148.9 (C), 164.8 (C)

[0265] MS m/z (%): 245(M⁺ 62), 176 (41) 175 (100) 145 (36), 117 (11), 89(14).

[0266] m.p. 152.5°-153° C., yield 44.1%

[0267] 1-(3-trans-benzo-1,3-dioxol-5-ylacryloyl)morpholine (RV-B03)

[0268]¹H-NMR (CDCl₃) δ: 7.61 (d, 1H, J=15.3, CH═CH), 6.73 (d, 1H,J=15.3, CH═CH—), 7.03 (d,1H J=1.4, Ar-7H), 6.80 (d,1H J=8.0, Ar-8H),7.01 (d, d, 1H J=1.4, 8.0 Ar-9H), 5.99 (s, 2H, O—CH₂—O), 3.72 (br, 4H,CH₂—N—CH₂ (morpholine)), 3.67 (br, 4H, CH₂—O—CH₂(morpholine)),

[0269]¹³C-NMR (CDCl₃): 42.6 (CH₂), 46.2 (CH₂), 66.8 (CH₂), 46.5 (CH₂),101.4 (CH₂), 106.3 (CH), 108.5 (CH), 114.4 (CH), 123.9 (CH), 129.5 (CH),143.0 (CH) 148.2 (C), 148.9 (C), 149.1 (C), 165.6 (C)

[0270] MS m/z (%): 261(M⁺ 60), 176 (24) 175 (100) 145 (30), 117 (10), 89(11).

[0271] m.p. 160°-160.3° C., yield 50.1%

[0272] 5. Synthesis of 3-trans-benzo-1,3-dioxol-5-ylacrylic acid methylester (RV-BB1)

[0273] To 3,4-methylenedioxycinnamic acid (2 g, 0.01 mol,1 eq) methanol(4 ml, 10 eq) was added. Sulphuric acid (0.2 ml) was added and refluxedovernight. The solvent was rotary evaporated to yield solid residue.This residue was dissolved in ether and washed with water (2×100 ml) and5% NaHCO₃ (3×100 ml) and with water (2×100 ml). The organic fraction wasdried over anhydrous sodium sulphate and rotary evaporated to yieldwhite solid. Recrystallisation from ethyl acetate/petroleum spirityielded crystals (69.4% yield) m.p. 133.70-134.2° C. (Lit m.p. 134° C.)⁸

[0274] 3-trans-benzo-1,3-dioxol-5-ylacrylic acid methyl ester (RV-BB1)

[0275]¹H-NMR (CDCl₃) δ: 7.59 (d, 1H, J=15.9, CH═CH), 6.26 (d, 1H,J=15.9, CH═CH—),7.03 (d,1H J=1.5, Ar-7H), 6.81 (d,1H J=8.0, Ar-8H), 7.01(d, d, 1H J=1.5, 8.0 Ar-9H), 6.00 (s, 2H, O—CH₂—O), 3.79 (s, 3H, OCH₃)

[0276]¹³C-NMR (CDCl₃): 51.6 (CH₃), 101.5 (t CH₂), 106.5 (CH), 108.5(CH), 115.7 (CH), 124.4 (CH), 128.8 (CH), 144.5 (CH) 148.3 (C), 148.6(C), 148.2 (C), 167.6 (C)

[0277] MS m/z (%): 206(M⁺ 100), 175 (68) 175 (100) 145 (27), 117 (10),89 (11).

References

[0278]⁷H. Staudinger and H. Schneider. (1923). Chem. Ber. 56, 699.

[0279]⁸Takemoto et al. (1985). Chemical and Pharmaceutical Bulletin 23,1161.

[0280] 6. Synthesis of tetrahydropiperine (RV-C02)

[0281] Piperine (2 g, 7 mmol) was hydrogenated in ethanol (50 ml) over5% Pd—C under a pressure of hydrogen at 10 psi for 30 mins to givetetrahydropiperine (1.59 g, 78% yield) as an oil².

[0282] Tetrahydropiperine (RV-C02)

[0283]¹H-NMR (CDCl₃) δ: 2.55 (t, 4H, J=7.0 CH²⁻CH⁻²CH²⁻CH₂), 2.32 (t,4H, J=7.0 CH₂CH²⁻CH²⁻CH₂) 6.66 (d,1H J=1.3, Ar-7H), 6.70 (d,1H J=8.0,Ar-10H), 6.61 (d, d, 1H J=1.2, 8.0 Ar-11H), 5.89 (s, 2H, O—CH₂—O), 3.53(t, 2H, N—CH₂ (piperidine)) 3.35 (t, 2H, N—CH₂ (piperidine)) 1.63 (m,2H, CH²⁻CH²⁻CH₂ (piperidine)) 1.54 (m, 2H, CH²⁻CH²⁻CH²⁻(piperidine))

[0284]¹³C-NMR (CDCl₃): 24.5 (CH₂), 24.9 (CH₂), 25.5 (CH₂), 26.5 (CH₂),31.4 (CH₂), 33.2 (CH₂), 35.4 (CH₂), 42.5(CH₂), 46.6(CH₂), 100.7 (CH₂),108.0 (CH), 108.8 (CH), 109.0 (CH), 121.0 (C), 145.4 (C) 147.4 (C),171.1 (C)

[0285] MS m/z (%): 289 (M⁺ 71), 204 (31), 154 (23), 148 (22), 141 (23),140 (38), 135 (28) 127 (100), 112 (23), 86 (12), 84 (24), 70 (10), 36(11)

[0286] Preparation of 5-(3,4-methylenedioxy phenyl)-pentanoic acidcyclohexyl amide (RV-C04)

[0287] To 5-(3,4-methylenedioxy phenyl)-2E,4E-pentadienoic acidcyclohexyl amide (300 mg) was added 5% Pd/C (30 mg) and hydrogenated thecontents at 30 psi for 1 hr. The solution was filtered and rotaryevaporated to yield a white solid. Recrystallisation from ethylacetateand petroleum spirit yielded pure white crystals (255 mg, yield 84%).m.p. 145.4° C.-146.3° C.

[0288] Preparation of 7-(3,4-methylenedioxy phenyl)-heptanoic acidpiperidine amide (RV-C-05)

[0289] To 7-(3,4-methylenedioxy phenyl)-2E,4E,6E-heptatrienoic acidpiperidine amide (150 mg, 0.06 mmole) was added 5% Pd/C (15 mg) andhydrogenated the contents at 30 psi for 30 min to give7-(3,4-methylenedioxy phenyl)-heptanoic acid piperidine amide as an oil.

[0290]¹H-NMR (CDCl₃) δ: 6.65 (d,1H J=1.6, Ar-7-H), 6.71 (d,1H J=7.8,Ar-10-H), 6.60 (d, d, 1H J=1.6, 8.0 Ar-11-H), 5.90 (s, 2H, O—CH₂—O),5.43 (s, 1H, NH), 2.53 (t, 2H, J=7.7 (CH₂—CH²⁻CH₂CH₂)) 2.14 (t, 2H,J=7.7 ((CH²⁻CH²⁻CH²⁻CH₂)) 1.62-1.91 (m, 10H, CH₂—CH²⁻CH²⁻CH₂,CH²⁻CH²⁻CH₂ (cyclohexyl amide) 1.07-1.30 (m, 4H, CH²⁻CH⁻CH₂(cyclohexylamide))

[0291]¹³C-NMR (CDCl₃): 25.3 ((CH₂)₂), 25.7 (CH₂),25.9 (CH₂), 31.3 (CH₂),31.7 (CH₂), 33.6 (CH₂), 35.8 (CH₂), 37.3 (CH₂), 48.4 (CH), 101.1 (CH₂),108.4 (CH), 109.2 (CH), 121.4 (CH), 136.4 (C), 145.8 (C), 147.8 (C),172.2 (C),

[0292] MS m/z (%): 303 (M⁺ 98), 204 (72), 176 (13), 168(16), 162 (12)161 (14), 154 (27), 148 (66), 141 (61) 135 (100) 74 (24) 60 (60)

[0293]¹H-NMR (CDCl₃) δ: 6.66 (d,1H J=1.5, Ar-7-H), 6.71 (d,1H J=7.8,Ar-10-H), 6.60 (d, d, 1H J=1.6, 8.0 Ar-11-H), 5.90 (s, 2H, O—CH₂—O),3.53 (t, 2H, J=5.4 CH²⁻N⁻CH₂) 3.37 (t, 2H, J=5.7, (CH₂—N—CH₂) 2.51 (t,2H, J=7.7 (CH₂—CH²⁻CH²⁻CH²⁻CH²⁻CH₂)) 2.33(t, 2H, J=7.7((CH₂—CH²⁻CH²⁻CH²⁻CH²⁻CH₂)) 1.52-1.65 (m, 10H, hydrocarbon CH₂, CH₂,CH₂—CH₂—CH₂ (Piperidine)) 1.34 (m, 4H, CH₂ CH₂)

[0294]¹³ C-NMR (CDCl₃): 24.9 (CH₂), 25.8 (CH₂),25.9 (CH₂), 26.9 (CH₂),29.3(CH₂), 29.7 (CH₂), 31.3 (CH₂), 31.9 (CH₂), 33.8 (CH₂), 42.9 (CH₂),47.1 (CH₂), 101.8 (CH₂), 108.4 (CH), 109.2 (CH), 121.4 (CH), 137.0 (C),145.7 (C), 147.8 (C), 171.8 (C),

[0295] MS m/z (%): 317 (M⁺ 78), 232 (11), 204 (10), 183 (30), 182 (15),154 (21) 148 (43), 141 (41), 127 (100), 112 (43), 85 (49)

[0296] Yield 51.2%

[0297] 7. Synthesis of 3-benzo-1,3-dioxol-5-ylpropionic acid piperidide

[0298] 7.1 Synthesis of 3-benzo-1,3-dioxol-5-ylpropionic acid

[0299] 3-benzo-1,3-dioxol-5-ylacrylic acid (2 g) was hydrogenated inethanol (50 ml) over 5% Pd—C under a pressure of hydrogen at 10 psi for40 mins to give 3-benzo-1,3-dioxol-5-ylpropionic acid (1.67 g, 80%yield) as a solid, m.p. 86.1°-88.3° C. (Lit m.p. 87-88° C.)¹⁰

[0300] 7.2 Synthesis of 3-benzo-1,3-dioxol-5-ylpropionic acid piperidide(RV-C03)

[0301] The method was adapted from that reported for piperlonguminine(section 2.2) but utilising 3-benzo-1,3-dioxol-5-ylpropionic acid andpiperidine as the acid and amine components respectively. A mixture of3-benzo-1,3-dioxol-5-ylpropionic acid (200 mg, 0.0026 mole, 1 eq) andtriethylamine (0.27 ml, 0.002 mole, 2 eq) in dichloromethane (50 ml) wasstirred for 15 min at 0° C. To this mixture methanesulfonyl chloride(0.11 ml, 0.0015 mole, 1.5 eq) was added and stirred for further 30 minat 0° C. Piperidine (0.15 ml, 0.0015 mole, 1.5 eq) was added to themixture and stirred for 1 h at 0° C. and 1 h at room temperature.Dichloromethane (50 ml) was added to the mixture which was then washedwith 5% HCl (3×100 ml), saturated aqueous NaHCO₃ (3×100 ml) and water(3×100 ml). The organic fraction was dried over anhydrous sodiumsulphate, filtered and rotary evaporated to yield brown oil (65% yield).

[0302] 3-benzo-1,3-dioxol-5-ylpropionic acid piperidide (RV-C03)

[0303]¹H-NMR (CDCl₃) δ: 2.87 (t, 2H, J=7.3 CH₂), 2.57 (t, 2H, J=7.0CH²⁻CH₂) 6.70 (d,1H J=1.5, Ar-7H), 6.72 (d,1H J=8.0, Ar-10H), 6.66 (d,d, 1H J=1.2, 8.0 Ar-11H), 5.90 (s, 2H, O—CH₂—O), 3.55 (t, 2H, N—CH₂(piperidine)) 3.34 (t, 2H, N—CH₂ (piperidine)) 1.62 (m, 2H, CH²⁻CH²⁻CH₂(piperidine)) 1.49 (m, 2H, CH²⁻CH²⁻CH₂(piperidine))

[0304]¹³C-NMR (CDCl₃): 25.7 (CH₂), 25.9 (CH₂), 26.6 (CH₂), 31.7 (CH₂),35.8 (CH₂), 43.1(CH₂), 47.1(CH₂), 101.2 (CH₂), 109.2 (CH), 109.3 (CH),121.5 (CH), 135.6 (C), 146.2 (C) 148.0 (C), 170.8 (C)

References

[0305]⁹Biswanath Das., A. Kasinatham., and P. Madhusudhan. (1998).Regioselective reduction of αβ-double bond of some naturally occuringdienamides using NABH₄/I₂ system. Tetrahedron Letters 39, 677-678.

[0306]¹⁰Perkin, Robinson, (1907) Journal of Chemical Society 91, 1084

[0307] 8 Synthesis of amide derivatives of methoxy-substituted cinnamicacid

[0308] A mixture of monomethoxycinnamic acid (200 mg, 0.89 mmol, 1 eq)and triethylamine (2.4 ml, 1.78 mmol, 2 eq) in dichloromethane (50 ml)was stirred for 15 min at 0° C. To this mixture methanesulfonyl chloride(1.02 ml, 1.33 mmol, 1.5 eq) was added and stirred for further 30 min at0° C. Piperidine (0.23 ml, 1.33 mmol, 1.5 eq) was added to the mixtureand stirred for 1 h at 0° C. and 1 h at room temperature. Thendichloromethane (50 ml) was added to the mixture, which was then washedwith 5% HCl (3×100 ml), saturated aqueous NaHCO₃ (3×100 ml) and water(3×100 ml). The organic fraction was dried over anhydrous sodiumsulphate, filtered and rotary evaporated to yield an oil. This oil waspurified by chromatography on silica gel using ethyl acetate/petroleumspirit (2:8) as an eluant.

[0309] The piperidine amide of 3,4 dimethoxycinnamic acid was preparedin the same way utilising 200 mg of the acid.

[0310] 1-(2-methoxy-cinnamoyl)-piperidine (RV-G01)

[0311]¹H-NMR (CDCl₃) δ: 7.56 (d, 1H, CH═CH), 7.29 (d, 1H, J=7.8 ArH),7.12(d, 1H, J=7.6 ArH) 7.0 (d,d 1H, J=1.8 ArH) 6.86-6.90 (m,ArH), 6.88(d, 1H, J=15.4 CH═CH), 3.58-3.66 (br, 4H, CH₂—N—CH₂ (piperidine))1.56-1.71 (m, 6H, CH²⁻CH₂CH₂ (piperidine)) 3.83 (s, 3H, OCH₃)

[0312]¹³C-NMR (CDCl₃): 25.7 (CH₂), 26.0 (CH₂), 27.1 (CH₂), 43.7(CH₂),47.4(CH₂), 55.7(CH₃), 113.4 (CH),115.3 (CH), 118.5 (CH),120.6 (CH),130.1(CH),142.4 (CH), 137.3 (C), 160.2 (C), 165.6 (C)

[0313] MS m/z (%): 245(M⁺ 28), 162 (22), 161 (100), 133 (20), 118 (24),113 (14), 84 (51) yield 25.5%

[0314] 1-(3-methoxy-cinnamoyl)piperidine (RV-G02)

[0315]¹H-NMR (CDCl₃) δ: 7.60 (d, 1H, J=15.4, CH═CH), 7.29 (d, 1H, J=7.8ArH), 7.12(d, 1H, J=7.6 ArH) 7.0 (d,d 1H, J=1.8 ArH) 6.86-6.90 (m,ArH),6.88 (d, 1H, J=15.4 CH═CH), 3.58-3.66 (br, 4H, CH₂—N—CH₂ (piperidine))1.56-1.71 (m, 6H, CH²⁻CH₂CH₂ (piperidine)) 3.83 (s, 3H, OCH₃)

[0316]¹³C-NMR (CDCl₃): 25.7 (CH₂), 26.0 (CH₂), 27.1 (CH₂), 43.7(CH₂),47.4(CH₂), 55.7(CH₃), 113.4 (CH),115.3 (CH), 118.5 (CH),120.6 (CH),130.1(CH),142.4 (CH), 137.3 (C), 160.2 (C), 165.6 (C)

[0317] MS m/z (%): 245(M⁺ 77), 162 (65), 161 (100), 133 (20), 118 (24),113 (14), 84 (51)

[0318] m.p. 68°-70° C., yield 31.4%

[0319] 1-(4-methoxy-cinnamoyl)piperidine (RV-G03)

[0320]¹H-NMR (CDCl₃) δ: 7.61 (d, 1H, J=15.4, CH═CH), 7.47 (d, 2H, J=7.8ArH), 6.87-6.90 (m,2H,ArH), 6.77 (d, 1H, J=15.4 CH═CH), 3.58-3.65 (br,4H, CH₂—N—CH₂ (piperidine)) 1.52-1.69 (m, 6H, CH²⁻CH₂CH₂ (piperidine))3.82 (s, 3H, OCH₃)

[0321]¹³C-NMR (CDCl₃): 25.6 (CH₂), 26.0 (CH₂), 26.4 (CH₂), 43.7(CH₂),47.4(CH₂), 55.7(CH₃), 114.5 (CH),115.6 (CH), 118.5 (CH),121.9 (CH),129.6(CH),142.2 (CH), 132.8 (C), 161.0 (C), 166.0 (C)

[0322] MS m/z (%): 245(M⁺ 71), 162 (17), 161 (100), 133 (26), 118 (12),113 (14), 84 (24), 77 (36)

[0323] 1-(3,4-dimethoxycinnamoyl)piperidine (RV-G04)

[0324]¹H-NMR (CDCl₃) 60 MHz δ: 7.61 (1H, CH═CH), 7.23 (1H, ArH),6.98(1H, ArH) 6.82 (1H, J=1.8 ArH) 6.68 (1H, CH═CH), 3.58-3.65 (br, 4H,CH₂—N—CH₂ (piperidine)) 1.5-1.8 (6H, CH²⁻CH₂CH₂ (piperidine)) 3.91 (s,6H, OCH₃)₂)

[0325] MS m/z (%): 275(M⁺ 62), 192 (48), 191 (100), 161 (18), 118 (11),84 (26), 77 (12), yield 42.3%

1. A method of treating a subject having a skin condition selected fromthe group of conditions (a) those treatable by stimulation of melanocyteproliferation and (b) melanomas, which comprises administering to thesubject an effective amount of a compound of formula (1)

wherein n =0 or 1; p is 0 or 1; q is 0 or 1 when n=p=q=0, R³ and R⁴represent hydrogen or together represent a carbon to carbon double bond;when n=0 and one of p and q=1, R³ and R⁴ together and one of R⁵ and R⁶together or R⁷ and R⁸ together represent carbon to carbon double bonds,R³ and R⁴ together represent a carbon to carbon double bond and R⁵ andR⁶ or R⁷ and R⁸ represent hydrogen atoms, R³ and R⁴ represent hydrogenand one of R⁵ and R⁶ together or R⁷ and R⁸ together represent carbon tocarbon double bonds or R³, R⁴, R⁵, R⁶, R⁷ and R⁸ all represent hydrogenatoms; when n=0 and p=q=1, R³ and R⁴ together and one of R⁵ and R⁶together or R⁷ and R⁸ together represent carbon to carbon double bondsthe other of R⁵, R⁶, R⁷ and R₈ representing hydrogen, R³ and R⁴ togetherrepresent a carbon to carbon double bond and R⁵ and R⁶ or R⁷ and R⁸represent hydrogen atoms, R³ and R⁴ represent hydrogen and one of R⁵ andR⁶ together or R⁷ and R⁸ together represent carbon to carbon doublebonds the other of R⁵, R⁶, R⁷ and R⁸ representing hydrogen, R³ and R⁴together, R⁵ and R⁶ together and R⁷ and R⁸ together represent carbon tocarbon double bonds or R³, R⁴, R⁵, R⁶, R⁷ and R⁸ all represent hydrogenatoms; or optionally when n is 1 R² and R³ together represent a carbonto carbon double bond and one or more of R⁴ and R⁵ together, R⁵ and R⁶together, R⁶ and R⁷ together or R⁷ and R⁸ together represent a carbon tocarbon double bond the other of R⁴ to R⁸ representing hydrogen; m=1, 2or 3; when m=1, R¹ represents an alkoxy group having from 1 to 3 carbonatoms or a hydroxy group; when m=2, each R¹ independently represents analkoxy group having from 1 to 3 carbon atoms or the two R¹s togetherrepresent a 3′,4′-methylenedioxy group; when m=3, two R¹s togetherrepresent a 3′,4′-methylenedioxy group and the other R¹ represents analkoxy group having from 1 to 3 carbon atoms or a hydroxy group; R⁹represents a pyrrolidino, piperidino, 4-methylpiperidino or morpholinogroup, a N-monoalkylamino group of 4 to 6 carbon atoms, aN-monocycloalkylamino group of 4 to 7 carbon atoms, a3′,4′-methylenedioxy-substituted benzylamino or 2-phenethylamino groupor R⁹ represents an alkoxy group of 1 to 6 carbon atoms; in any of itsE, Z geometrically isomeric forms.
 2. The method of claim 1, wherein thesubject is a patient suffering from a melanoma.
 3. The method of claim1, wherein the subject is a patient suffering from a skin disordertreatable by stimulation of melanocyte proliferation.
 4. The method ofclaim 1, wherein the skin disorder is vitiligo.
 5. The method of claim1, wherein the compound is administered topically to the area of theskin to be treated.
 6. The method of claim 1, wherein the compound offormula (1) is one in which: n=0, one of p and q=1, R³ and R⁴ togetherand one of R⁵ and R⁶ together or R⁷ and R⁸ together represent the secondbond of a carbon to carbon double bond the other of R⁵, R⁶, R⁷ and R⁸representing hydrogen, m=2, the R¹ groups together represent3′,4′-methylenedioxy and R⁹ represents a pyrrolidino, piperidino,morpholino, cyclohexylamino or isobutylamino group.
 7. The method ofclaim 6, wherein the compound is of the E, E geometric configuration. 8.The method of claim 1, wherein the compound of formula (I) is one inwhich n is 0, one of p and q is 1, R³, R⁴, R⁵, R⁶, R⁷ and R⁸ representhydrogen and R⁹ is cyclohexylamino
 9. The method of claim 1, wherein thecompound of formula (1) is piperine, being the E, E-isomer of thecompound of formula (1) in which n=0, one of p or q=1, R³ and R⁴together and one of R⁵ and R⁶ together or R⁷ and R⁸ together representthe second bond of a carbon to carbon double bond, the other of R⁵, R⁶,R⁷ and R⁸ representing hydrogen, m=2, the R¹ groups together represent3′,4′-methylenedioxy and R⁹ represents piperidino, and the geometricconfiguration is E, E.
 10. A method of treating a subject having a skincondition selected from the group of conditions (a) those treatable bystimulation of melanocyte proliferation and (b) melanomas, whichcomprises administering to the subject an effective amount of a compoundof formula (1)

in which (a) n is 0, p and q are each 0 or 1, m is 2, the R¹s togetherrepresent a 3′,4′-methylenedioxy group, R² and R³, together with thecarbon atoms to which they are attached form a carbon to carbon doublebond and, when p and q are each 0 or 1, R⁵ and R⁶ and R⁷ and R⁸ togetherwith the carbon atoms to which they are attached, form a carbon tocarbon double bond and R⁹ is piperidino, or (b) n is 0, one of p or q is1 and (i) m is 3, the R¹s being 3′,4′-methylenedioxy and 6′-methoxy or(ii) m is 2, the R¹s being 3′-hydroxy-4′-methoxy; or (iii) m is 1 andthe R¹ is 4′-hydroxy; and R³to R⁹ are as defined in case (a) above, or(c) n is 0, one of p and q is 1, R⁹ is piperidino, pyroolidino,isobutylamino or methoxy and all other symbols are as defined in case(a) above, or (d) n is 0, one of p and q is 1, R⁴ and R⁵ representhydrogen atoms and either R² and R³ also do or R² and R³ together withthe carbon atoms to which they are attached form a carbon to carbondouble bond; and m, R¹ and R⁹ are as defined in case (a) above; (e) n is0, p q=1 and R³, R⁴, R⁵, R⁶, R⁷ and R⁸ represent hydrogen; (f) n is 0,one of p and q is 1, R³, R⁴, R⁵, R⁶, R⁷ and R⁸ represent hydrogen and R⁹is cyclohexylamino; and in all of which cases (a) to (f) the molecule isin the E,E or all E geometric configuration or in case (a) when n is 1may be in the Z,Z, Z,E or E,Z geometric configuration.
 11. The method ofclaim 10, wherein the subject is a patient suffering from a melanoma.12. The method of claim 10, wherein the subject is a patient sufferingfrom a skin disorder treatable by stimulation of melanocyteproliferation.
 13. The method of claim 10, wherein the skin disorder isvitiligo.
 14. A compound of formula (I)

in which n is 0, one of p and q is 1, R³, R⁴, R⁵, R⁶, R⁷ and R⁸represent hydrogen and R⁹ is cyclohexylamino